U.S. patent application number 14/214922 was filed with the patent office on 2014-09-18 for wound healing compositions and treatments.
This patent application is currently assigned to CoDa Therapeutics, Inc.. The applicant listed for this patent is CoDa Therapeutics, Inc.. Invention is credited to Scott BANNAN, Bradford DUFT, David EISENBUD, Grove MATSUOKA, Anthony PHILLIPS, David POOL, Tracey SUNDERLAND.
Application Number | 20140275218 14/214922 |
Document ID | / |
Family ID | 51529997 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140275218 |
Kind Code |
A1 |
PHILLIPS; Anthony ; et
al. |
September 18, 2014 |
WOUND HEALING COMPOSITIONS AND TREATMENTS
Abstract
This invention concerns improved methods, uses, and kits for
treating chronic wounds through the administration of anti-connexin
agents, particularly anti-connexin 43 antisense polynucleotides.
The methods, uses, and kits of the invention are based on the
surprising and unexpected discovery that chronic wounds that do not
increase or decrease in size by more than a pre-determined amount
during a pre-treatment phase are more amenable to successful
treatment than wounds whose size varies outside the target range
during the pre-treatment phase.
Inventors: |
PHILLIPS; Anthony; (San
Diego, CA) ; EISENBUD; David; (San Diego, CA)
; BANNAN; Scott; (San Diego, CA) ; POOL;
David; (San Diego, CA) ; MATSUOKA; Grove; (San
Diego, CA) ; SUNDERLAND; Tracey; (San Diego, CA)
; DUFT; Bradford; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CoDa Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
CoDa Therapeutics, Inc.
San Diego
CA
|
Family ID: |
51529997 |
Appl. No.: |
14/214922 |
Filed: |
March 15, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13844762 |
Mar 15, 2013 |
|
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14214922 |
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Current U.S.
Class: |
514/44A ;
514/44R; 536/23.5; 600/587 |
Current CPC
Class: |
A61P 17/02 20180101;
A61K 9/06 20130101; A61B 5/4848 20130101; A61K 9/70 20130101; A61B
5/0064 20130101; A61K 31/7105 20130101; A61B 5/445 20130101; A61P
43/00 20180101; A61B 5/4839 20130101; A61B 5/7275 20130101; A61K
48/00 20130101; A61P 29/00 20180101; A61K 9/0014 20130101; A61K
31/7088 20130101; A61K 47/00 20130101; C12N 2310/14 20130101; C12N
2320/35 20130101; A61B 2503/40 20130101; A61K 47/38 20130101; C07K
14/47 20130101; A61B 5/0077 20130101; A61B 5/1109 20130101; C12N
15/1138 20130101; A61K 47/10 20130101; A61B 5/1079 20130101; C12N
2320/30 20130101; C12N 2310/11 20130101; A61P 3/10 20180101; A61B
5/1072 20130101 |
Class at
Publication: |
514/44.A ;
514/44.R; 536/23.5; 600/587 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61B 5/00 20060101 A61B005/00; A61B 5/107 20060101
A61B005/107 |
Claims
1. A method of treating a refractory wound, comprising: measuring
the size a skin wound upon initial presentation for treatment to
obtain a first size measurement; administering standard of care
such as compression bandaging and/or off-loading to the wound;
measuring the size of the wound after 2-4 weeks of administering
the standard of care to obtain a second size measurement;
determining that the second size indicator of the wound is within a
predetermined range from about -30 to about +35% of the first size
measurement, thereby identifying a refractory wound; and
administering to the refractory wound a pharmaceutical composition
comprising a pharmaceutical carrier suitable for topical
administration of an anti-connexin polynucleotide to a connexin
selected from connexin 26, connexin 30 and connexin 43.
2. A method of treating a refractory venous leg ulcer, comprising
administering to the ulcer a pharmaceutical carrier suitable for
topical administration and about 3-30 mg/mL of an anti-connexin
polynucleotide to a connexin selected from connexin 26, connexin 30
and connexin 43.
3. An article of manufacture for use in treating a refractory
wound, comprising a receptacle containing a composition comprising
a pharmaceutical carrier suitable for topical administration having
about 20-23% of a nonionic polyoxyethylene-polyoxypropylene and
about 3-30 mg/mL of an anti-connexin oligodeoxynucleotide to a
connexin selected from connexin 26, connexin 30 and connexin 43,
and, and instructions for the treatment of the refractory wound, by
topically administering the composition to or in the proximity of
the wound.
4. A method of detecting a refractory wound with an increased
likelihood of complete closure following topical administration to
the recalcitrant wound of a composition comprising a pharmaceutical
carrier suitable for topical administration of an anti-connexin
oligodeoxynucleotide to a connexin selected from connexin 26,
connexin 30 and connexin 43, the method comprising: measuring the
size of a skin wound upon initial presentation for treatment, to
obtain a first size measurement; administering standard of care,
such as compression bandaging and/or off-loading to the wound;
measuring the size of the skin wound after about 2-4 weeks of
administering the standard of care, to obtain a second size
measurement; detecting that the second size measurement is within
-30% to +35% of the first size measurement, thereby detecting a
refractory wound having an increased likelihood of complete closure
following topical administration to the recalcitrant wound of a
pharmaceutical composition comprising a pharmaceutical carrier
suitable for topical administration having about 20-23% of a
nonionic polyoxyethylene-polyoxypropylene and about 3-30 mg/mL of
an anti-connexin oligodeoxynucleotide to a connexin selected from
connexin 26, connexin 30 and connexin 43; and administering the
pharmaceutical composition to the wound.
5. The method of any of claims 1-4 wherein measuring the first or
second size indicator further comprises using planimetry,
digitizing techniques, stereophotogrammetry, a ruler, wound
tracing, a handheld laser scanner or a camera.
6. A method according to claim 5 wherein the chronic wound is
selected from the group consisting of a venous ulcers, venous
stasis ulcers, arterial ulcers, pressure ulcers, diabetic ulcers,
diabetic foot ulcers, vasculitic ulcers, decubitus ulcers, burn
ulcers, trauma-induced ulcers, infectious ulcers, mixed ulcers, and
pyoderma gangrenosum.
7. A method according to claim 1 wherein the chronic wound is a
venous leg ulcer.
8. A method according to claim 1 comprising a plurality of
administrations of the pharmaceutical composition.
9. A method according to claim 8 wherein the pharmaceutical
composition is applied repeatedly until wound closure is
achieved.
10. A method according to claim 9 wherein the administrations are
periodic.
11. A method according to claim 10 wherein the periodic
administrations occur at regularly scheduled intervals, optionally,
daily, every other day, twice weekly, weekly, twice monthly, and
monthly.
12. A method according to claim 11 wherein the periodic
administrations occur once weekly until wound closure is
achieved.
13. A method according to claim 1 wherein the anti-connexin 43
agent is an anti-connexin 43 polynucleotide.
14. A method according to claim 1 wherein the anti-connexin 43
polynucleotide, optionally an oligodeoxynucleotide or modified
oligodeoxynucleotide comprising from about 18 to about 32
nucleotides.
15. A method according to claim 1 wherein the subject is a mammal,
optionally a human.
16. A method according to claim 1 wherein the run-in period ranges
from about 14 days to about 28 days.
17. A kit for treating a refractory wound, comprising a container
containing a therapeutic amount of an anti-connexin 43 agent and
instructions for use of the anti-connexin 43 agent in performing a
method according to claim 1.
18. The method of claims 1-4 wherein the pharmaceutical composition
comprises a pharmaceutically acceptable carrier and an
anti-connexin 43 polynucleotide present at a concentration selected
from the following: from about 10-200 .mu.M, 200-300 .mu.M, 300-400
.mu.M, 400-500 .mu.M, 500-600 .mu.M, 600-700 .mu.M, 700-800 !AM,
800-900 .mu.M, 900-1000 or 1000-1500 .mu.M, or 1500 .mu.M-2000
.mu.M, 2000 .mu.M-3000 .mu.M, 3000 .mu.M-4000 .mu.M, 4000
.mu.M-5000 .mu.M, 5000 .mu.M-6000 .mu.M, 6000 .mu.M-7000 .mu.M,
7000 .mu.M-8000 .mu.M, 8000 .mu.M-9000 .mu.M, 9000 .mu.M-10,000
.mu.M, 10,000 .mu.M-11,000 .mu.M, 11,000 .mu.M-12,000 .mu.M, 12,000
.mu.M-13,000 .mu.M, 13,000 .mu.M-14,000 .mu.M, 14,000 .mu.M-15,000
.mu.M, 15,000 .mu.M-20,000 .mu.M, 20,000 .mu.M-30,000 .mu.M, or
from about 30,000 .mu.M-50,000 .mu.M.
19. The method according to claim 13 wherein the anti-connexin 43
polynucleotide is selected from: an oligodeoxynucleotide, a
modified oligodeoxynucleotide, an unmodified oligodeoxynucleotide,
an antisense polynucleotide, an unmodified antisense
polynucleotide, and a modified antisense polynucleotide.
20. The method of claim 13 wherein the anti-connexin 43
polynucleotide is a sequence selected from SEQ ID NOS: 1-3, or a
sequence having up to about 100 nucleotides of a sequence
complementary to SEQ.ID.NO.134 or SEQ.ID.NO.135.
21. The method of claim 13 wherein the anti-connexin 43
polynucleotide is a sequence selected from SEQ ID NOS: 1 and 2.
22. The method of claim 13 wherein the anti-connexin 43
polynucleotide is an antisense polynucleotide having at least about
70 percent homology with SEQ ID NOS: 1 and 2.
23. The method according to any of claims 1-13 wherein the
anti-connexin 43 polynucleotide is an antisense polynucleotide that
hybridizes to connexin 43 mRNA under conditions of medium to high
stringency.
24. The method according to claim 23, wherein said antisense
polynucleotide having a sequence selected from SEQ.ID.NO:1-3, or a
sequence having up to about 40 nucleotides of a sequence
complementary to SEQ.ID.NO.134 or SEQ.ID.NO.135.
25. The method according to claim 23, wherein said antisense
polynucleotide is selected from: TABLE-US-00003 (SEQ ID NO: 1) GTA
ATT GCG GCA AGA AGA ATT GTT TCT GTC; and (SEQ ID NO: 2) GTA ATT GCG
GCA GGA GGA ATT GTT TCT GTC (SEQ ID NO: 3) GGC AAG AGA CAC CAA AGA
CAC TAC CAG CAT.
26. The method according to claim 23, wherein said antisense
polynucleotide has from about 15 to about 35 nucleotides and is
sufficiently complementary to connexin 43 mRNA to form a duplex
having a melting point greater than 20.degree. C. under
physiological conditions.
27. The method according to claim 23, wherein the antisense
polynucleotide has from about 15 to about 35 nucleotides and has at
least about 70 percent homology to an antisense sequence of
connexin 43 mRNA.
28. The method according to claim 13, wherein said anti-connexin
agent is an RNAi or siRNA polynucleotide.
29. The method according to any of claims 1-16 or 18-28 which is
formulated as a gel.
30. The method according to claim 13, wherein the pharmaceutical
formulation is administered topically.
31. The method according to one of claims 13 or 30, wherein said
gel is a polyoxyethylene-polyoxypropylene copolymer-based gel or a
carboxymethylcellulose-based gel.
32. The method according to one of claims 13 or 30, wherein said
gel is a pluronic gel.
33. The method according to one of claims 13 or 30 according to
claim 67 or 85, wherein said gel is a pluronic F-127.
34. The method according to one of claims 13 or 30, wherein the
pharmaceutically acceptable carrier comprises an alginate.
35. The method according to one of claims 13 or, wherein the
pharmaceutically acceptable carrier comprises a hydrogel.
36. The method according to one of claims 13 or, 30, wherein the
pharmaceutically acceptable carrier comprises a hydrogel selected
from the group consisting of hydrogels containing a cellulose
derivative and hydrogels containing polyacrylic acid.
37. The method according to one of claims 13 or 30, wherein the
pharmaceutically acceptable carrier is a cellulose-based
carrier.
38. The method according to one of claims 13 or 30, wherein the
pharmaceutically acceptable carrier comprises a cellulose-based
carrier selected from the group consisting of hydroxyethyl
cellulose, hydroxymethyl cellulose, carboxymethyl cellulose,
hydroxypropylmethyl cellulose and mixtures thereof.
39. The method according to one of claims 13 or 30, wherein the
composition is formulated for sustained release.
40. The method according to one of claims 13 or 30, wherein the
composition is formulated for slow release, extended release, or
controlled release.
41. The method according to one of claims 13 or 30, wherein the
composition is a cream, ointment, emulsion, lotion, spray, salve,
foam or paint.
42. A kit or an article of manufacture comprising package material
containing a composition for use in the method of claim 13 together
with instructions for use in treating a refractory chronic
wound.
43. An article of manufacture according to claim 42 wherein the
wound is characterized at least in part by increased expression of
connexin 43.
44. An article of manufacture according to claim 42 wherein the
wound is characterized at least in part by inflammation.
45. An article of manufacture according to claim 42 wherein the
wound is a dehiscent wound.
46. An article of manufacture according to claim 42 wherein the
wound is a venous ulcer.
47. An article of manufacture according to claim 42 wherein the
wound is a diabetic ulcer.
48. An article of manufacture according to claim 42 wherein the
wound is a diabetic foot ulcer.
49. An article of manufacture according to claim 42 wherein the
wound is a pressure ulcer.
50. An article of manufacture according to claim 42 wherein the
wound is an arterial ulcer.
51. An article of manufacture according to claim 42 wherein the
wound is a vasculitic ulcer.
52. An article of manufacture according to claim 42 wherein the
wound is a skin ulcer resulting from trauma or a burn.
53. An article of manufacture according to claim 42 wherein the
subject is diabetic.
54. An article of manufacture according to claim 42 wherein the
subject has a venous valve malfunction and/or venous
insufficiency.
55. An article of manufacture according to claim 42 wherein the
subject has an arterial blockage.
56. An article of manufacture according to claim 42 wherein said
composition is applied more than once.
57. An article of manufacture according to claim 42 wherein the
instructions provide for administration of said composition about
once per week.
58. An article of manufacture according to claim 42 wherein the
instructions provide for bi-weekly administration of said
composition.
59. An article of manufacture according to claim 42 wherein the
instructions provide for administration of said composition every
3-7 days.
60. An article of manufacture according to claim 42 wherein the
instructions state that a repeat application of said composition
may be made in the event that wound healing slows or is
stalled.
61. An article of manufacture according to claim 42 wherein the
subject is a human.
62. An article of manufacture according to claim 42, wherein the
subject is a non-human animal.
63. A method of treating a subject having a wound not healing at an
expected rate, comprising administering to the wound a composition
comprising an anti-connexin 43 polynucleotide and a
pharmaceutically acceptable carrier, wherein the amount of said
anti-connexin 43 polynucleotide administered to said wound ranges
from about 30 to about 500 .mu.g per square centimeter of said
wound, the improvement comprising determining whether the wound
heals by more than about 30-35% during a 2-4 week run-in period
during which the wound is treated by compression and, if not,
applying said anti-connexin 43 composition to said wound.
64. A program of instructions tangibly embodied on a non-transitory
computer-readable medium for execution by a computer to perform a
wound measuring process in a planimetry, stereophotogrammetry,
computer, tablet, or PDA, or handheld laser device for measuring a
size indicator of a wound, the computer having a processor
configured to determine whether a chronic wound has an increased
likelihood of complete closure, the program providing: receipt, on
the computer, of measurements of a size indicator of the wound
before a first treatment is administered; receipt, on the computer,
of measurements of the size indicator of the wound after a 2-4 week
run-in period; comparing the size indicator measurements from steps
a) and b) and detecting that the area of the wound is within -15%
to +30% of its area prior to administering the first treatment,
thereby determining an increased likelihood of complete closure
following topical administration to the recalcitrant wound of a
composition comprising from about 20-23% of a nonionic
polyoxyethylene-polyoxypropylene copolymer and a single-stranded
anti-connexin 43 antisense oligodeoxynucleotide present at a
concentration from 3.0 to about 500.0 milligrams per milliliter.
Description
RELATED APPLICATION
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 13/844,762, filed Mar. 15, 2013, the contents of which are
herein incorporated by reference as if set forth in their
entirety.
FIELD OF THE INVENTION
[0002] The inventions relate to methods of identifying and treating
refractory chronic wounds that do not heal at expected rates, and
dose regimens and articles of manufacture comprising gap junction
modulators useful for treating those wounds.
BACKGROUND
[0003] The following includes information that may be useful in
understanding the present invention. It is not an admission that
any of the information provided herein is prior art, or relevant,
to the presently described or claimed inventions, or that any
publication or document that is specifically or implicitly
referenced is prior art.
[0004] In humans and other mammals, wound injury triggers an
organized complex cascade of cellular and biochemical events that
will in most cases result in a healed wound. An ideally healed
wound is one that restores normal anatomical structure, function,
and appearance at the cellular, tissue, organ, and organism levels.
Wound healing, whether resulting from trauma, microbes, or foreign
materials, proceeds via a complex process encompassing a number of
overlapping phases, including inflammation, epithelialization,
angiogenesis, and matrix deposition. Normally, these processes lead
to a mature wound and a certain degree of scar formation. Although
inflammation and repair mostly occur along a prescribed course, the
sensitivity of the process is dependent on the balance of a variety
of wound healing modulating factors, including, for example, a
complex network of regulatory cytokines and growth factors.
[0005] Gap junctions are cell membrane structures that facilitate
direct cell-cell communication. A gap junction channel is formed of
two connexons (hemichannels), each composed of six connexin
subunits. Each hexameric connexon docks with a connexon in the
opposing membrane to form a single gap junction. Gap junction
channels are reported to be found throughout the body.
[0006] Connexins are a family of proteins, commonly named according
to their molecular weight or classified on a phylogenetic basis
into alpha, beta, and gamma subclasses. At least 20 human and 19
murine isoforms have been identified. Different tissues and cell
types are reported to have characteristic patterns of connexin
protein expression.
[0007] Antisense technology has been reported for the modulation of
the expression for genes implicated in viral, fungal, and metabolic
diseases. See, e.g., U.S. Pat. Nos. 5,166,195, 5,004,810. Antisense
technology has also been developed to modulate connexins and treat
wounds. See, e.g., U.S. Pat. Nos. 7,098,190, 7,879,811, 7,902,164,
7,919,474, 8,034,789, 8,059,486, 8,063,023, 8,181,580, and
8,314,074. Peptide inhibitors of gap junctions and hemichannels
have also been reported. See, e.g., WO2006/134494, published U.S.
patent application publication no. 20100279921.
[0008] Despite advances in the understanding of the principles
underlying the wound healing process, there remains a significant
unmet need in suitable therapeutic options for chronic wound care.
These inventions address this continuing need.
BRIEF SUMMARY
[0009] The inventions described and claimed herein have many
attributes and embodiments including, but not limited to, those set
forth or described or referenced in this summary section, which is
not intended to be all-inclusive. The inventions described and
claimed herein are not limited to or by the features or embodiments
identified in this summary section, which is included for purposes
of overview illustration only and not limitation.
[0010] This disclosure relates to methods of identifying and
treating refractory chronic wounds, and dose regimens and articles
of manufacture useful for treating those wounds. Pharmaceutical
compositions comprising the articles of manufacture, and useful in
the methods disclosed herein comprise effective doses of
anti-connexin polynucleotides (e.g., a connexin antisense
oligodeoxynucleotide such as a single-stranded anti-connexin
oligodeoxynucleotide) to connexin 26, connexin 30 and/or connexin
43, as well as other connexins as disclosed herein.
[0011] As used herein, in one embodiment of the invention, and by
way of example, refractory wounds are chronic wounds, or wounds
that do not heal at expected rates, such as delayed-healing wounds
and incompletely healing wounds, which do not decrease in size by
more than about 30% (+30%) over a standard-of-care treatment period
using, for example, compression bandaging or off-loading devices,
over about two to four weeks and which do not increase in wound
size by more than 15% (-15%). This standard-of-care treatment
period may also be referred to as a "run-in" or "pretreatment"
period. The increase or decrease in wound size may be referred to
broadly as "surface area reduction" (also referred to as SAR"). The
SAR range during run-in, e.g., -15% to +30%, may be referred to as
the run-in SAR range. Upon presentation of a chronic wound for
treatment, standard-of-care is provided for a two to four week
period. Estimates and/or formal measurements of the size of the
wound upon presentation, and at the end of the run-in period are
used to determine if the wound is refractory to standard of care
treatment by assessing whether it falls with a designated SAR
run-in range. In some aspects, standard of care during the run-in
period is multi-layer compression bandaging, for example. In other
aspects, the standard of care is off-loading, for example. In some
embodiments, the standard of care treatment during the run-in
period is single-layer compression bandaging, or a compression
stocking. Compression bandages are typically changed or reapplied
approximately once per week. In other embodiments, the standard of
care treatment during the run-in period is reduction of pressure,
or offloading. Foot pressures, shock, and shear may be reduced with
appropriately fitted shoes, insoles, and socks. Total non-weight
bearing using a wheelchair or crutches is another effective method
of relieving pressure. Other off-loading standard of care
modalities include total contact casts, removal casts, and
removable cast walkers.
[0012] In other embodiments, refractory wounds are chronic wounds,
or wounds that do not heal at expected rates, such as
delayed-healing wounds and incompletely healing wounds, which do
not decrease in size by more than about 30% (+30%) over a
standard-of-care treatment period using compression bandaging over
about two to four weeks.
[0013] In still other embodiments, refractory wounds are chronic
wounds, or wounds that do not heal at expected rates, such as
delayed-healing wounds and incompletely healing wounds, which do
not decrease in size by more than about 35% (+35%) over a
standard-of-care treatment period using compression bandaging which
do not increase in wound size by more than 15% (-15%).
[0014] In further embodiments, refractory wounds are chronic
wounds, or wounds that do not heal at expected rates, such as
delayed-healing wounds and incompletely healing wounds, which do
not increase or decrease in size by more than about -5%/+30%,
-10%/+30%, -15%/+30%, -20%/+30%, 25%/+30% or -30%/+30%, over a
standard-of-care treatment period using compression bandaging over
about two to four weeks.
[0015] It has been surprisingly observed that the refractory
chronic wounds are susceptible to administration of pharmaceutical
compositions comprising a pharmaceutically acceptable carrier
suitable for topical administration and, for example, from about
0.5 mg/mL to about 40 mg/mL, or by way of additional example from
about 1 to 30 mg/mL, of an anti-connexin 26, anti-connexin 30 or
anti-connexin 43 polynucleotide.
[0016] Accordingly, in one aspect, this disclosure relates to a
method of treating a refractory wound, the method comprising:
[0017] determining a size of a chronic wound upon initial
presentation for treatment to obtain a first size measurement or
estimation;
[0018] administering standard-of-care, such as, for example,
compression bandaging and/or off-loading, to the wound;
[0019] determining the size of the wound about 2-4 weeks after
administering said standard-of-care to obtain a second size
measurement;
[0020] determining that the second size indicator of the wound is
within a predetermined range (for example, -15 to +30%/-35% to
+30%) of the first size measurement, or that it has not healed by
more a predetermined amount (e.g., a wound SAR of more than about
+30-35%) thereby identifying a refractory wound; and
[0021] administering at or in proximity to the wound a
pharmaceutical composition comprising an effective amount of an
anti-connexin 43 polynucleotide, for example. Examples of suitable
doses, dose amounts and dose concentrations and formulations are
described herein.
[0022] The size of the wound may be determined by various methods
known in the art, including, for example, by a measurement or
reasonable estimation or approximation of any physical dimension of
the wound, such as surface area, length of the longest diameter
and/or length of the longer perpendicular bisector to the longest
diameter. For deep wounds with a relatively small surface area,
such as a diabetic foot ulcers, volume may also be used as the size
measurement. Other known wound measurement methods include
planimetry, wound tracing, digitizing techniques, and
stereophotogrammetry, as well as simple ruler-based methods. Three
commercially available wound measurement techniques include the
Visitrak system (Smith and Nephew Healthcare, Hull, U.K.), a
digital photography and image processing system (Analyze, version
6.0; AnalyzeDirect, Lenexa, Kans.), and an elliptical measurement
method using the standard formula (nab) for the calculation of the
area of an ellipse.
[0023] In another aspect, this disclosure relates to methods of
detecting a refractory chronic wounds susceptible to treatment by
pharmaceutical compositions comprising, for example, from about 0.5
mg/mL to about 40 mg/mL or from about 1 to 30 mg/mL of, for
example, an anti-connexin 43 polynucleotide, the method
comprising
[0024] measuring a size indicator of a wound upon initial
presentation for treatment to obtain a first size measurement;
[0025] administering standard-of-care, such as, for example,
compression bandaging and/or off-loading, to the wound;
[0026] measuring the area of the wound about 2-4 weeks after
administering initiating standard of care treatment to obtain a
second size measurement;
[0027] determining that the second size indicator of the wound is
within a predetermined range of the first size measurement as
described herein, thereby detecting a slowly progressing or
refractory wound susceptible to treatment by administration of a
pharmaceutical composition comprising a therapeutically effective
amount of, for example, an anti-connexin 43 polynucleotide. Other
connexin targets are contemplated as described herein.
[0028] In another aspect, this disclosure relates to kits, packages
and/or articles of manufacture useful in treating a subject having
a refractory wound, comprising a receptacle containing a
pharmaceutical composition comprising an anti-connexin 43 antisense
oligodeoxynucleotide present at a therapeutically effective amount
or concentration, e.g., from 1.0 to 3.0 to about 30.0 to 100
milligrams per milliliter, and a pharmaceutically acceptable
carrier, e.g., a nonionic polyoxyethylene-polyoxypropylene
copolymer; and instructions for use of the compositions as
described and claimed herein. Such medicaments include those for
the treatment of a subject as described herein.
[0029] In another aspect this invention relates to a method of
detecting a refractory wound with an increased likelihood of
complete closure following topical administration to the wound of a
composition comprising a nonionic polyoxyethylene-polyoxypropylene
copolymer, e.g., poloxamer 407 (at a concentration, for example, of
about 15-30%, such as 25-27%) and a single-stranded anti-connexin
43 antisense oligodeoxynucleotide, for example, present at a
concentration from about 3.0 to about 30 milligrams per milliliter,
or from about 1.0 to about 100 milligrams per milliliter, for
example, the method comprising:
[0030] measuring a size indicator of a wound upon initial
presentation for treatment to obtain a first size measurement;
[0031] administering standard-of-care, such as, for example,
compression bandaging and/or off-loading, to the wound;
[0032] measuring the size indicator of the wound about 2-4 weeks
after initiating the standard of care treatment to obtain a second
size measurement; and
[0033] detecting that the area of the wound is within a
predetermined size range, for example from about -15% to +30% of
its size after the run-in period, thereby determining an increased
likelihood of complete closure following topical administration to
the refractory wound of a composition comprising said anti-connexin
43 antisense oligodeoxynucleotide.
[0034] In some embodiments, the pharmaceutical composition
comprises an effective amount of a 3-30 mg/mL or 1-100 mg/mL
anti-connexin 43 polynucleotide or other suitable doses disclosed
herein. The pharmaceutical composition may further comprise one or
more pharmaceutical carriers suitable for topical administration.
In one embodiment, the pharmaceutical composition may comprise
about 20-30% nonionic polyoxyethylene-polyoxypropylene copolymer,
and/or other pharmaceutical carriers disclosed herein.
[0035] In one aspect the anti-connexin polynucleotide may be
present in the pharmaceutical composition at a concentration of
from about 3.0 to about 30 mg/mL, or from about 1.0 to about 100
mg/mL. In some aspects, the anti-connexin polynucleotide may be
present at a concentration of about 3, about 5, about 10, or about
30 mg/ml, or any amount up to about 100 mg/mL. In other aspects the
anti-connexin polynucleotide may be present in the pharmaceutical
composition at a concentration of from about 100 .mu.M to about
5000 .mu.M, for example. In some embodiments, a therapeutically
effective amount of a composition of the invention comprises a
pharmaceutically acceptable carrier and an anti-connexin agent such
as a single-stranded anti-connexin 43 oligodeoxynucleotide present
at a concentration from about 0.5 to about 40 mg per milliliter
(mL; mg/mL) or from about 3 to about 30 mg/mL, or from about 100
.mu.M to about 5000 .mu.M.
[0036] One or more doses may be administered to a subject having a
refractory wound. In some embodiments, one or more doses of the
pharmaceutical composition may be repeatedly administered at
appropriate or desired intervals. In some embodiments, the
pharmaceutical composition may be administered repeatedly, for
example, daily, or one to six times per week. For example, the
anti-connexin agent-containing compositions and formulations
described herein can be administered once per week until healing is
seen to be proceeding or is complete, as desired. Compositions of
the invention may also be applied more frequently, for example, two
or three times/week. They may also be applied biweekly, or monthly.
The frequency of administration and dose may change over the course
of treatment as the wound area and volume change. In addition,
further application(s) can be made in the event wound healing once
again becomes stalled or delayed.
[0037] According to another aspect of the present invention, wound
re-epithelialization and/or formation of granulation tissue is
promoted. Methods of promoting re-epithelialization of skin wounds
comprise administering to a subject having a wound that is not
healing at the expected rate, including, for example, a delayed
healing or an incompletely healing wound or a chronic wound, an
anti-connexin agent, e.g., an anti-connexin polynucleotide, in an
amount effective to promote re-epithelialization. Analogous methods
can be used to regulate epithelial basal cell division and
growth.
[0038] It has been surprisingly observed that the refractory wounds
are susceptible to administration of pharmaceutical compositions
comprising from 0.5 to 40 mg/mL anti-connexin polynucleotide, for
example, or higher concentrations, at or in proximity to the wound
a pharmaceutical composition comprising and a pharmaceutically
acceptable carrier suitable for topical administration. Detecting
refractory wounds may advantageously be used to identify patients
amenable to treatment with pharmaceutical compositions comprising
from 0.5 to 40 mg/mL anti-connexin polynucleotide, e.g., from 3 to
30 mg/mL of anti-connexin 43 ODN, by way of example. Other suitable
doses and dose concentrations are provided herein.
[0039] In some aspects of the methods of this invention, as noted
above, the size of a patient's wound is assessed by ascertaining
length, width, depth, edge circumference, volume, or surface area
of the wound, or function thereof. Any suitable method may be used,
including direct manual measurement, a laser scanner, an imaging
device such as a camera, computer tablet or PDA, surface mapping,
etc. The currently preferred metric for wound size is wound surface
area, preferably obtained as the product of wound length multiplied
by wound width, wound tracing, or by planimetry.
[0040] Size changes during the run-in period surprisingly and
unexpectedly have been discovered to range from an increase in
wound size by not more than about 30% to a decrease in wound size
by not more than 15% during the run in period, for example, and
other ranges and/or threshold amounts as previously noted.
Preferred run-in phases range from about 7 to about 30 days,
preferably from about 7 days to about 21 days, even more preferably
from about 7 days to about 14 days. A 14-day run-in is
preferred.
[0041] These and other aspects of the present inventions, which are
not limited to or by the information in this Brief Summary, are
provided below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIGS. 1A-C show the results from a Phase 2B clinical study
using a -40% to +40% wound size change during the run in period
following a 10 week treatment period. FIG. 1A shows the plot for %
complete wound closure the patients in each study arm: standard of
care (SOC), vehicle alone, and low dose (1 mg) or high dose (1 mg)
of an exemplary anti-connexin 43 polynucleotide. FIG. 1B shows the
plot for wound surface area reduction for patients in each study
arm. FIG. 1C shows that patients selected based on the -40% to +40%
wound size change in the run in period did not exhibit
statistically significant responses in the context of the surface
area reduction endpoint and the complete wound closure secondary
endpoint.
[0043] FIG. 2 shows the plot of % complete wound closure at week 10
from a re-analysis of the data from the Phase 2B study a patient
subpopulation in each arm of the study that met -15% to +30% wound
size change criteria.
[0044] FIG. 3 shows the plot of surface area reduction based on the
re-analysis of the data from the Phase 2B study for patient
subpopulation that met -15% to +30% wound size change criteria in
each arm of the study.
[0045] FIG. 4 shows that the patient subpopulation that met the
-15% to +30% wound size change criteria exhibited statistically
significant (P<0.05) responses in the context of the surface
area reduction endpoint and the complete wound closure secondary
endpoint.
DETAILED DESCRIPTION
[0046] Wounds that do not heal at expected rates, including
slow-healing wounds, delayed-healing wounds, incompletely healing
wounds, dehiscent wounds, and chronic wounds, often result in
infection and can lead to amputation or death. Cell-cell
communication through the gap junctions plays pivotal roles in
wound healing. It has been discovered that use of certain
compounds, namely anti-connexin agents, including those described
or referenced herein or otherwise now known or later developed, can
block, inhibit, or alter cell communication to promote closure and
healing in wounds that do not heal at expected rates, including
slow-healing wounds, delayed-healing wounds, incompletely healing
wounds, dehiscent wounds, and chronic wounds. Furthermore, as
described herein, it has surprisingly and unexpectedly been
discovered that healing of refractory chronic wounds by
administration of one or more anti-connexin agents to connexin 26
(Cx26), connexin 30 (Cx30) or connexin 43 (Cx43), for example, can
be further promoted by administering a desired anti-connexin agents
to a patient whose chronic wound(s) remain within a certain size
range during a standard of care (pre-treatment) run-in phase. Other
connexin targets include connexin 30.3 (Cx30.3), connexin 31
(Cx31), connexin 31.1 (Cx31.1), connexin 32 (Cx32), connexin 37
(Cx37), connexin 40 (Cx40), and connexin 45 (Cx45), i.e., is an
anti-connexin 26, 30, 30.3, 31, 31.1, 32, 37, 40, 43, or 45
polynucleotide. Preferably, during a pretreatment phase of from
about 1 to about 30 days, preferably from about 5 days to about 20
days, even more preferably from about 7 days to about 14 days, the
wound to be treated does not increase in size by more than about
30%, for example, or increase in size by more than about 35% as
another example, or increase in size by more than about 30% or
decrease in size by more than about 15%, for example.
[0047] It has been surprisingly observed that the refractory wounds
that do not increase in wound size by about 30% or decrease in
wound size by 15% are susceptible to administration of
pharmaceutical compositions comprising from about 0.5 mg/mL to
about 40 mg/mL, or from about 1 to 30 mg/mL anti-connexin
polynucleotide at or in proximity to the wound a pharmaceutical
composition comprising and a pharmaceutically acceptable carrier
suitable for topical administration. In other embodiments,
refractory wounds are chronic wounds, or wounds that do not heal at
expected rates, such as delayed-healing wounds and incompletely
healing wounds, which do not decrease in size by more than about
30% (+30%) over a standard-of-care treatment period using, for
example, compression bandaging or off-loading devices over about
two to four weeks. In still other embodiments, refractory wounds
are chronic wounds, or wounds that do not heal at expected rates,
such as delayed-healing wounds and incompletely healing wounds,
which do not decrease in size by more than about 35% (+35%) over a
standard-of-care treatment period using, for example, compression
bandaging or off-loading devices, which do not increase in wound
size by more than 15% (-15%). In further embodiments, refractory
wounds are chronic wounds, or wounds that do not heal at expected
rates, such as delayed-healing wounds and incompletely healing
wounds, which do not increase or decrease in size by more than
about -5%/+30%, -10%/+30%, -15%/+30%, -20%/+30%, 25%/+30% or
-30%/+30%, over a standard-of-care treatment period using, for
example, compression bandaging or off-loading devices, over about
two to four weeks. In other embodiments, the standard of care
treatment during the run-in period is reduction of pressure, or
offloading. Foot pressures, shock, and shear may be reduced with
appropriately fitted shoes, insoles, and socks. Total non-weight
bearing using a wheelchair or crutches is another effective method
of relieving pressure. Other off-loading standard of care
modalities include total contact casts, removal casts, and
removable cast walkers.
[0048] In some embodiments, a therapeutically effective amount of a
composition of the invention comprises a pharmaceutically
acceptable carrier and an anti-connexin agent such as a
single-stranded anti-connexin 43 oligodeoxynucleotide present at a
concentration from about 0.5 to about 40.0 mg per milliliter (mL;
mg/mL) or from about 3 to about 30 mg/mL. Preferred concentrations
range from about 1.5 to about 30 milligrams per milliliter (mg/mL),
about 1.5 to about 10 mg/mL, or about 3, about 5, about 10, or
about 30 mg/ml. In some aspects the total dose of anti-connexin
polynucleotide administered may be about 100 .mu.g to about 30
mg.
[0049] Particularly preferred concentrations for connexin antisense
polynucleotides (e.g., single-stranded anti-connexin 43
oligodeoxynucleotides) range from about 150 .mu.M to about 10,000
.mu.M. Viewed another way, a refractory chronic skin wound can be
effectively treated in accordance with the invention by
administering about 150 .mu.g to about 10,000 mg of a connexin
antisense polynucleotide (e.g., a single-stranded anti-connexin 43
oligodeoxynucleotide) per square centimeter of wound surface
area.
DEFINITIONS
[0050] As used herein, a "disorder" is any disorder, disease, or
condition that would benefit from an agent that promotes healing of
chronic or delayed healing wounds which are refractory to standard
of care treatment, and/or reduces scar formation when such wounds
are treated. For example, included are wound-associated
abnormalities in connection with neuropathic, ischemic, and
microvascular pathology; pressure over bony area [tailbone
(sacral), hip (trochanteric), buttocks (ischial), or heel of the
foot]; reperfusion injury; and conditions associated with valve
reflux etiology and related conditions.
[0051] As used herein, "subject" refers to any mammal, including
humans, domestic and farm animals, and zoo, sports, or pet animals,
such as dogs, horses, cats, sheep, pigs, cows, etc. The preferred
mammal herein is a human, including adults, children, and the
elderly.
[0052] As used herein, "preventing" means preventing in whole or in
part, or ameliorating or controlling.
[0053] As used herein, a "therapeutically effective amount" in
reference to the compounds or compositions of the instant invention
refers to the amount sufficient to induce a desired biological,
pharmaceutical, or therapeutic result. That result can be
alleviation of the signs, symptoms, or causes of a disease or
disorder or condition, or any other desired alteration of a
biological system. In the present invention, the result will
involve the promotion and/or improvement of wound healing,
including rates of wound healing and closure of wounds, in whole or
in part. Other benefits include decreases in swelling, inflammation
and/or scar formation, in whole or in part.
[0054] As used herein, the term "treating" refers to both
therapeutic treatment and prophylactic or preventative
measures.
[0055] As used herein, "simultaneously" is used to mean that the
one or more anti-connexin agents (e.g., an anti-connexin
polynucleotide, e.g., an antisense polynucleotide) are administered
concurrently, whereas the term "in combination" is used to mean
they are administered, if not simultaneously or in physical
combination, then "sequentially" within a timeframe that they both
are available to act therapeutically. Thus, administration
"sequentially" may permit one agent to be administered within
minutes (for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30) minutes or
a matter of hours, days, weeks or months after the other provided
that one or more anti-connexin polynucleotides are concurrently
present in effective amounts. The time delay between administration
of the components will vary depending on the exact nature of the
components, the interaction therebetween, and their respective
half-lives.
[0056] As used herein, an "anti-connexin agent" decreases or
inhibits expression of a connexin mRNA, pre-mRNA, and/or connexin
protein. Anti-connexin agents include anti-connexin polynucleotides
include, without limitation, antisense compounds such as antisense
polynucleotides, other polynucleotides (such as polynucleotides
having siRNA or ribozyme functions), peptidomimetics, and other
compounds that interfere with connexin protein activity, function,
transport, localization, etc. Suitable examples of an anti-connexin
polynucleotide include an antisense polynucleotide that targets a
connexin mRNA. Accordingly, suitable anti-connexin polynucleotides
include, for example, antisense polynucleotides (e.g., Cx43
antisense polynucleotides) that modulate expression or activity of
connexins and gap junctions in selected tissues, cells, and
subjects.
[0057] The term "wound dressing" refers to a dressing for topical
application to a wound and excludes compositions suitable for
systemic administration. For example, the one or more anti-connexin
agents (such as an anticonnexin polynucleotide) may be dispersed in
or on a solid sheet of wound contacting material such as a woven or
nonwoven textile material, or may be dispersed in a layer of foam
such as polyurethane foam, or in a hydrogel such as a polyurethane
hydrogel, a polyacrylate hydrogel, gelatin, carboxymethyl
cellulose, pectin, alginate, and/or hyaluronic acid hydrogel, for
example in a gel or ointment. In certain embodiments the one or
more anti-connexin agents are dispersed in or on a biodegradable
sheet material that provides sustained release of the active
ingredients into the wound, for example a sheet of freeze-dried
collagen, freeze-dried collagen/alginate mixtures (available under
the Registered Trade Mark FIBRACOL from Johnson & Johnson
Medical Limited) or freeze-dried collagen/oxidized regenerated
cellulose (available under the Registered Trade Mark PROMOGRAN from
Johnson & Johnson Medical Limited).
[0058] As used herein, "wound promoting matrix" includes for
example, synthetic or naturally occurring matrices such as
collagen, acellular matrix, crosslinked biological scaffold
molecules, tissue based bioengineered structural framework,
biomanufactured bioprostheses, and other implanted structures such
as for example, vascular grafts suitable for cell infiltration and
proliferation useful in the promotion of wound healing. Additional
suitable biomatrix material may include chemically modified
collagenous tissue to reduces antigenicity and immunogenicity.
Other suitable examples include collagen sheets for wound
dressings, antigen-free or antigen reduced acellular matrix
(Wilson, et al. (1990), Trans Am Soc Artif Intern 36:340-343), or
other biomatrices that have been engineered to reduce the antigenic
response to the xenograft material. Other matrices useful in
promotion of wound healing may include for example, processed
bovine pericardium proteins comprising insoluble collagen and
elastin (Courtman, et al. (1994), J Biomed Mater Res 28:655-666)
and other acellular tissue which may be useful for providing a
natural microenvironment for host cell migration to accelerate
tissue regeneration (Malone J M et al. (1984) J Vasc Surg
1:181-91). The invention contemplates a synthetic or natural matrix
comprising one or more anti-connexin agents described herein.
[0059] As used herein, the term "wound" includes an injury to any
tissue, including for example, delayed or difficult to heal wounds,
and chronic wounds. Examples of wounds may include both open and
closed wounds. The term "wound" may also include for example,
injuries to the skin and subcutaneous tissue initiated in different
ways (e.g., pressure sores from extended bed rest and wounds
induced by trauma) and with varying characteristics. Wounds may be
classified into one of four grades depending on the depth of the
wound: i) Grade I wounds limited to the epithelium; ii) Grade II
wounds extending into the dermis; iii) Grade III wounds extending
into the subcutaneous tissue; and iv) Grade IV (or full-thickness
wounds) wounds wherein bones are exposed (e.g., a bony pressure
point such as the greater trochanter or the sacrum).
[0060] The term "partial thickness wound" refers to wounds that
encompass Grades I-III. Examples of partial thickness wounds
include pressure sores, venous stasis ulcers, and diabetic ulcers.
The present invention contemplates treating all wounds of a type
that do not heal at expected rates, including, delayed-healing
wounds, incompletely healing wounds, and chronic wounds.
[0061] "Wound that does not heal at the/an expected rate" means an
injury to any tissue, including delayed or difficult to heal wounds
(including delayed or incompletely healing wounds), and chronic
wounds. Examples of wounds that do not heal at the expected rate
include ulcers, such as diabetic ulcers, diabetic foot ulcers,
vasculitic ulcers, arterial ulcers, venous ulcers, venous stasis
ulcers, pressure ulcers, decubitus ulcers, infectious ulcers,
trauma-induced ulcers, burn ulcers, ulcerations associated with
pyoderma gangrenosum, and mixed ulcers. Other wounds that do not
heal at expected rates include dehiscent wounds.
[0062] As used herein, a "delayed" or "difficult to heal" wound may
include, for example, a wound that is characterized at least in
part by 1) a prolonged inflammatory phase, 2) a slow forming
extracellular matrix, and/or 3) a decreased rate of
epithelialization or closure.
[0063] The term "chronic wound" generally refers to a wound that
has not healed. Wounds that do not heal within three months, for
example, are considered chronic. Chronic wounds include venous
ulcers, venous stasis ulcers, arterial ulcers, pressure ulcers,
diabetic ulcers, diabetic foot ulcers, vasculitic ulcers, decubitus
ulcers, burn ulcers, trauma-induced ulcers, infectious ulcers,
mixed ulcers, and pyoderma gangrenosum. The chronic wound may be an
arterial ulcer that comprises ulcerations resulting from complete
or partial arterial blockage. The chronic wound may be a venous or
venous stasis ulcer that comprises ulcerations resulting from a
malfunction of the venous valve and the associated vascular
disease. In certain embodiments a method of treating a chronic
wound is provided where the chronic wound is characterized by one
or more of the following AHCPR stages of pressure ulceration: stage
1, stage 2, stage 3, and/or stage 4.
[0064] As used herein, chronic wound may refer to, for example, a
wound that is characterized at least in part by one or more of (1)
a chronic self-perpetuating state of wound inflammation, (2) a
deficient and defective wound extracellular matrix, (3) poorly
responding (senescent) wound cells especially fibroblasts, limiting
extracellular matrix production, and/or (4) failure of
re-epithelialization due in part to lack of the necessary
extracellular matrixorchestration and lack of scaffold for
migration. Chronic wounds may also be characterized by 1) prolonged
inflammation and proteolytic activity leading to ulcerative
lesions, including for example, diabetic, pressure (decubitous),
venous, and arterial ulcers; 2) progressive deposition of matrix in
the affected area, 3) longer repair times, 4) less wound
contraction, 5) slower re-epithelialization, and 6) increased
thickness of granulation tissue.
[0065] The term "refractory" chronic wound or "refractory" wound
refers to wounds that do not heal at expected rates, such as
delayed-healing wounds, incompletely healing wounds, and chronic
wounds, and which do not, for example, increase in wound size by
more than about 30% (are partially refractory to administration of
standard of care) and which do not decrease in wound size by more
than 15% (-15%) (are totally refractory to administration of
standard of care) during a two to four week run-in period.
Following presentation for treatment of a wound that does not heal
at the expected rate, the wound is pre-treated using standard of
care treatment using, for example, compression bandaging or
off-loading devices. In some embodiments the refractory wounds are
wounds that do not heal at expected rates and which do not increase
in wound size by more than about 30% and do not decrease in size by
more than about 20%, 25%, 30%, or 35% during the run-in period. In
other embodiments, refractory wounds are chronic wounds, or wounds
that do not heal at expected rates, such as delayed-healing wounds
and incompletely healing wounds, which do not decrease in size by
more than about 30% (+30%) over a standard-of-care treatment period
using, for example, compression bandaging or off-loading devices,
over about two to four weeks. In still other embodiments,
refractory wounds are chronic wounds, or wounds that do not heal at
expected rates, such as delayed-healing wounds and incompletely
healing wounds, which do not decrease in size by more than about
35% (+35%) over a standard-of-care treatment period using, for
example, compression bandaging or off-loading devices, which do not
increase in wound size by more than 15% (-15%). In further
embodiments, refractory wounds are chronic wounds, or wounds that
do not heal at expected rates, such as delayed-healing wounds and
incompletely healing wounds, which do not increase or decrease in
size by more than about -5%/+30%, -10%/+30%, -15%/+30%, -20%/+30%,
25%/+30% or -30%/+30%, over a standard-of-care treatment period
using, for example, compression bandaging or off-loading devices,
over about two to four weeks. In some aspects of this disclosure,
the refractory wound may be, for example, a refractory skin ulcer,
such as a venous leg ulcer, or diabetic foot ulcer. In yet another
aspect, the invention includes methods for treating a subject
having or suspected of having any diseases, disorders, and/or
conditions characterized in whole or in part by a chronic wound or
delayed or incomplete wound healing, or other wound that does not
heal at an expected rate. In some embodiments, the patient has a
diabetic ulcer, a diabetic foot ulcer, a vasculitic ulcer, a venous
ulcer, a venous stasis ulcer, an arterial ulcer, a pressure ulcer,
a decubitus ulcer, an infectious ulcer, a trauma-induced ulcer, a
burn ulcer, ulcerations associated with pyoderma gangrenosum, or a
mixed ulcer or ulcers.
[0066] In the context of the instant inventions, the anti-connexin
agents are preferably administered topically (at and/or around the
site to be treated). Suitably, the anti-connexin agents, e.g.,
anti-connexin antisense polynucleotides are combined with a
pharmaceutically acceptable carrier, vehicle or diluent to provide
a pharmaceutical composition.
[0067] Exemplary chronic wounds may include "pressure ulcers."
Exemplary pressure ulcers may be classified into 4 stages based on
AHCPR (Agency for Health Care Policy and Research, U.S. Department
of Health and Human Services) guidelines. A stage I pressure ulcer
is an observable pressure related alteration of intact skin whose
indicators as compared to the adjacent or opposite area on the body
may include changes in one or more of the following: skin
temperature (warmth or coolness), tissue consistency (firm or boggy
feel) and/or sensation (pain, itching). The ulcer appears as a
defined area of persistent redness in lightly pigmented skin,
whereas in darker skin tones, the ulcer may appear with persistent
red, blue, or purple hues. Stage 1 ulceration may include
nonblanchable erythema of intact skin and the heralding lesion of
skin ulceration. In individuals with darker skin, discoloration of
the skin, warmth, edema, induration, or hardness may also be
indicators of stage 1 ulceration. Stage 2 ulceration may be
characterized by partial thickness skin loss involving epidermis,
dermis, or both. The ulcer is superficial and presents clinically
as an abrasion, blister, or shallow crater. Stage 3 ulceration may
be characterized by full thickness skin loss involving damage to or
necrosis of subcutaneous tissue that may extend down to, but not
through, underlying fascia. The ulcer presents clinically as a deep
crater with or without undermining of adjacent tissue. Stage 4
ulceration may be characterized by full thickness skin loss with
extensive destruction, tissue necrosis, or damage to muscle, bone,
or supporting structures (e.g., tendon, joint capsule). In certain
embodiments a method of treating a chronic wound is provided where
the chronic wound is characterized by one or more of the following
AHCPR stages of pressure ulceration: stage 1, stage 2, stage 3,
and/or stage 4.
[0068] Exemplary chronic wounds may also include "decubitus
ulcers." Exemplary decubitus ulcers may arise as a result of
prolonged and unrelieved pressure over a bony prominence that leads
to ischemia. The wound tends to occur in patients who are unable to
reposition themselves to off-load weight, such as paralyzed,
unconscious, or severely debilitated persons. As defined by the
U.S. Department of Health and Human Services, the major preventive
measures include identification of high-risk patients; frequent
assessment; and prophylactic measures such as scheduled
repositioning, appropriate pressure-relief bedding, moisture
barriers, and adequate nutritional status. Treatment options may
include for example, pressure relief, surgical and enzymatic
debridement, moist wound care, and control of the bacterial load.
In certain embodiments a method of treating a chronic wound is
provided wherein the chronic wound is characterized by decubitus
ulcer or ulceration, which results from prolonged, unrelieved
pressure over a bony prominence that leads to ischemia.
[0069] Chronic wounds may also include "arterial ulcers." Chronic
arterial ulcers are generally understood to be ulcerations that
accompany arteriosclerotic and hypertensive cardiovascular disease.
They are painful, sharply marginated, and often found on the
lateral lower extremities and toes. Arterial ulcers may be
characterized by complete or partial arterial blockage, which may
lead to tissue necrosis and/or ulceration. Signs of arterial ulcer
may include, for example, pulselessness of the extremity; painful
ulceration; small, punctate ulcers that are usually well
circumscribed; cool or cold skin; delayed capillary return time
(briefly push on the end of the toe and release, normal color
should return to the toe in about 3 seconds or less); atrophic
appearing skin (for example, shiny, thin, dry); and loss of digital
and pedal hair. In certain embodiments a method of treating a
chronic wound is provided wherein the chronic wound is
characterized by arterial ulcers or ulcerations due to complete or
partial arterial blockage.
[0070] Exemplary chronic wounds may include "venous ulcers."
Exemplary venous ulcers are the most common type of ulcer affecting
the lower extremities and may be characterized by malfunction of
the venous valve. The normal vein has valves that prevent the
backflow of blood. When these valves become incompetent, the
backflow of venous blood causes venous congestion. Hemoglobin from
the red blood cells escapes and leaks into the extravascular space,
causing the brownish discoloration commonly noted. It has been
shown that the transcutaneous oxygen pressure of the skin
surrounding a venous ulcer is decreased, suggesting that there are
forces obstructing the normal vascularity of the area. Lymphatic
drainage and flow also plays a role in these ulcers. The venous
ulcer may appear near the medial malleolus and usually occurs in
combination with an edematous and indurated lower extremity; it may
be shallow, not too painful and may present with a weeping
discharge from the affected site. In certain embodiments a method
of treating a chronic wound is provided wherein the chronic wound
is characterized by venous ulcers or ulcerations due to malfunction
of the venous valve and the associated vascular disease. In certain
embodiments a method of treating a chronic wound is provided
wherein the chronic wound is characterized by arterial ulcers or
ulcerations due to complete or partial arterial blockage.
[0071] Exemplary chronic wounds may include "venous stasis ulcers."
Stasis ulcers are lesions associated with venous insufficiency are
more commonly present over the medial malleolus, usually with
pitting edema, varicosities, mottled pigmentation, erythema, and
nonpalpable petechiae and purpura. The stasis dermatitis and ulcers
are generally pruritic rather than painful. Exemplary venous stasis
ulcers may be characterized by chronic passive venous congestion of
the lower extremities results in local hypoxia. One possible
mechanism of pathogenesis of these wounds includes the impediment
of oxygen diffusion into the tissue across thick perivascular
fibrin cuffs. Another mechanism is that macromolecules leaking into
the perivascular tissue trap growth factors needed for the
maintenance of skin integrity. Additionally, the flow of large
white blood cells slows due to venous congestion, occluding
capillaries, becoming activated, and damaging the vascular
endothelium to predispose to ulcer formation. In certain
embodiments a method of treating a chronic wound is provided
wherein the chronic wound is characterized by venous ulcers or
ulcerations due to malfunction of the venous valve and the
associated vascular disease. In certain embodiments a method of
treating a chronic wound is provided wherein the chronic wound is
characterized by venous stasis ulcers or ulcerations due to chronic
passive venous congestion of the lower extremities and/or the
resulting local hypoxia.
[0072] Exemplary chronic wounds may include "diabetic ulcers."
Diabetic patients are prone to ulcerations, including foot
ulcerations, due to both neurologic and vascular complications.
Peripheral neuropathy can cause altered or complete loss of
sensation in the foot and/or leg. Diabetic patients with advanced
neuropathy lose all ability for sharp-dull discrimination. Any cuts
or trauma to the foot may go completely unnoticed for days or weeks
in a patient with neuropathy. It is not uncommon to have a patient
with neuropathy notice that the ulcer "just appeared" when, in
fact, the ulcer has been present for quite some time. For patients
of neuropathy, strict glucose control has been shown to slow the
progression of the disease. Charcot foot deformity may also occur
as a result of decreased sensation. People with "normal" feeling in
their feet have the ability to sense automatically when too much
pressure is being placed on an area of the foot. Once identified,
our bodies instinctively shift position to relieve this stress. A
patient with advanced neuropathy loses this ability to sense the
sustained pressure insult, as a result, tissue ischemia and
necrosis may occur leading to for example, plantar ulcerations.
Additionally, microfractures in the bones of the foot, if unnoticed
and untreated, may result in disfigurement, chronic swelling and
additional bony prominences. Microvascular disease is one of the
significant complications for diabetics, which may also lead to
ulcerations. In certain embodiments a method of treating a chronic
wound is provided wherein the chronic wound is characterized by
diabetic foot ulcers and/or ulcerations due to both neurologic and
vascular complications of diabetes.
[0073] Exemplary chronic wounds can include "traumatic ulcers."
Formation of traumatic ulcers may occur as a result of traumatic
injuries to the body. These injuries include, for example,
compromises to the arterial, venous or lymphatic systems; changes
to the bony architecture of the skeleton; loss of tissue
layers-epidermis, dermis, subcutaneous soft tissue, muscle or bone;
damage to body parts or organs and loss of body parts or organs. In
certain embodiments, a method of treating a chronic wound is
provided wherein the chronic wound is characterized by ulcerations
associated with traumatic injuries to the body.
[0074] Exemplary chronic wounds can include "burn ulcers",
including first degree burn (i.e. superficial, reddened area of
skin); second degree burn (a blistered injury site which may heal
spontaneously after the blister fluid has been removed); third
degree burn (burn through the entire skin and usually require
surgical intervention for wound healing); scalding (may occur from
scalding hot water, grease or radiator fluid); thermal (may occur
from flames, usually deep burns); chemical (may come from acid and
alkali, usually deep burns); electrical (either low voltage around
a house or high voltage at work); explosion flash (usually
superficial injuries); and contact burns (usually deep and may
occur from muffler tail pipes, hot irons and stoves). In certain
embodiments, a method of treating a chronic wound is provided
wherein the chronic wound is characterized by ulcerations
associated with burn injuries to the body.
[0075] Exemplary chronic wounds can include "vasculitic ulcers."
Vasculitic ulcers also occur on the lower extremities and are
painful, sharply marginated lesions, which may have associated
palpable purpuras and hemorrhagic bullae. The collagen diseases,
septicemias, and a variety of hematological disorders (e.g.,
thrombocytopenia, dysproteinemia) may be the cause of this severe,
acute condition.
[0076] Exemplary chronic wounds can include pyoderma gangrenosum.
Pyoderma gangrenosum occurs as single or multiple, very tender
ulcers of the lower legs. A deep red to purple, undermined border
surrounds the purulent central defect. Biopsy typically fails to
reveal a vasculitis. In half the patients it is associated with a
systemic disease such as ulcerative colitis, regional ileitis, or
leukemia. In certain embodiments, a method of treating a chronic
wound is provided wherein the chronic wound is characterized by
ulcerations associated with pyoderma gangrenosum.
[0077] Exemplary chronic wounds can include infectious ulcers.
Infectious ulcers follow direct innoculation with a variety of
organisms and may be associated with significant regional
adenopathy. Mycobacteria infection, anthrax, diphtheria,
blastomyosis, sporotrichosis, tularemia, and cat-scratch fever are
examples. The genital ulcers of primary syphilis are typically
nontender with a clean, firm base. Those of chancroid and granuloma
inguinale tend to be ragged, dirty, and more extravagant lesions.
In certain embodiments, a method of treating a chronic wound is
provided wherein the chronic wound is characterized by ulcerations
associated with infection.
[0078] As used herein, the term "dehiscent wound" refers to a
wound, usually a surgical wound, which has ruptured or split open.
In certain embodiments, a method of treating a wound that does not
heal at the expected rate is provided wherein the wound is
characterized by dehiscence.
[0079] Anti-Connexin Agents
[0080] Anti-Connexin Polynucleotides
[0081] Anti-connexin polynucleotides include connexin antisense
polynucleotides as well as polynucleotides which have
functionalities that enable them to downregulate connexin
expression (for example, by downregulation of mRNA transcription or
translation). In the case of downregulation, this has the effect of
reducing direct cell-cell communication by gap junctions at the
site at which connexin expression is down-regulated.
[0082] The inventions generally relate to the use of an
anti-connexin agent, preferably an anti-connexin polynucleotide,
including, for example, an anti-connexin oligodeoxynucleotide
(ODN), directed to a messenger RNA (mRNA) or precursor thereof that
codes a connexin protein. Representative anti-connexin
polynucleotides used in the methods and articles of manufacture of
this disclosure include connexin antisense polynucleotides, as well
as RNAi polynucleotides, siRNA polynucleotides, shRNA
polynucleotides, ribozymes, DNAzymes, and other anti-connexin
polynucleotides that target a connexin messenger RNA (mRNA) or
precursor thereof. Other anti-connexin agents within the scope of
the invention include peptidomimetics and connexin phosphorylation
agents.
[0083] In the context of anti-connexin polynucleotides, such
molecules are preferably single-stranded polynucleotides, although
under physiological conditions all or portions of such molecules
may include one or more partially or completely double-stranded
regions. Such polynucleotides include those having modified and/or
unmodified backbone, and can be produced recombinantly or synthetic
chemistry.
[0084] In another embodiment, the anti-connexin polynucleotide may
be an anti-connexin 43, anti-connexin 26 and anti-connexin 30
polynucleotides, for example, an ODN, such as a single-stranded
anti-connexin oligodeoxynucleotide to connexin 26, connexin 30 or
connexin 43. In some embodiments the anti-connexin polynucleotide
is an anti-connexin 43 polynucleotide. In one embodiment, the
anti-connexin polynucleotide is a connexin 43 antisense
oligodeooxynucleotide such as a single-stranded anti-connexin
oligodeoxynucleotide to connexin.
[0085] In certain other embodiments, the anti-connexin agent is an
anti-connexin polynucleotide that targets a connexin mRNA or
precursor thereof (i.e., pre-mRNA), particularly an mRNA or
pre-mRNA that codes for connexin 43 (Cx43), connexin 26 (Cx26),
connexin 37 (Cx37), connexin 30 (Cx30), connexin 30.3 (Cx30.3),
connexin 31 (Cx31), connexin 31.1 (Cx31.1), or connexin 32 (Cx32),
connexin 40 (Cx40), and connexin 45 (Cx45), i.e., is an
anti-connexin 43, 26, 37, 30, 30.3, 31, 31.1, 32, 40 or 45
polynucleotide.
[0086] Particularly preferred anti-connexin polynucleotides include
anti-connexin oligodeoxynucleotides such as anti-connexin 43
oligodeoxynucleotides. Preferred anti-connexin polynucleotides
contain from about 18 to about 32 polynucleotides.
[0087] Accordingly, in another aspect, the invention provides
formulations comprising at least one anti-connexin agent, e.g., a
connexin antisense polynucleotide, together with a pharmaceutically
acceptable carrier or vehicle. In one preferred form, such
formulations contain a connexin antisense polynucleotide that
targets a single connexin mRNA species, most preferably, connexin
43 mRNA or pre-mRNA. Alternatively, the formulation can contain an
anti-connexin agent, e.g., a connexin antisense polynucleotide,
that targets more than one connexin mRNA species, e.g., an mRNA
species that codes for Cx43 as well as an mRNA species that codes
for Cx30, Cx26, Cx37, Cx31.1, or Cx32 and others as noted
herein.
[0088] According to one aspect, the downregulation of connexin
expression may be based generally upon the antisense approach using
antisense polynucleotides (such as DNA or RNA polynucleotides), and
more particularly upon the use of antisense oligodeoxynucleotides
(ODN). These polynucleotides (e.g., ODN) target the connexin
protein (s) to be down-regulated. Typically the polynucleotides are
single stranded, but may be double stranded.
[0089] The antisense polynucleotide may inhibit transcription
and/or translation of a connexin. Preferably the polynucleotide is
a specific inhibitor of transcription and/or translation from the
connexin gene or mRNA, and does not inhibit transcription and/or
translation from other genes or mRNAs. The product may bind to the
connexin gene or mRNA either (i) 5' to the coding sequence, and/or
(ii) to the coding sequence, and/or (iii) 3' to the coding
sequence.
[0090] The antisense polynucleotide is generally antisense to a
connexin mRNA. Such a polynucleotide may be capable of hybridizing
to the connexin mRNA and may thus inhibit the expression of
connexin by interfering with one or more aspects of connexin mRNA
metabolism including transcription, mRNA processing, mRNA transport
from the nucleus, translation or mRNA degradation. The antisense
polynucleotide typically hybridizes to the connexin mRNA to form a
duplex which can cause direct inhibition of translation and/or
destabilization of the mRNA. Such a duplex may be susceptible to
degradation by nucleases.
[0091] The antisense polynucleotide may hybridize to all or part of
the connexin mRNA. Typically the antisense polynucleotide
hybridizes to the ribosome binding region or the coding region of
the connexin mRNA. The polynucleotide may be complementary to all
of or a region of the connexin mRNA. For example, the
polynucleotide may be the exact complement of all or a part of
connexin mRNA. However, absolute complementarity is not required
and polynucleotides which have sufficient complementarity to form a
duplex having a melting temperature of greater than about
20.degree. C., 30.degree. C., or 40.degree. C. under physiological
conditions are particularly suitable for use in the present
invention.
[0092] Thus the polynucleotide is typically a homologue of a
sequence complementary to the mRNA. The polynucleotide may be a
polynucleotide which hybridizes to the connexin mRNA under
conditions of medium to high stringency such as 0.03M sodium
chloride and 0.03M sodium citrate at from about 50.degree. C. to
about 60.degree. C.
[0093] For certain aspects, suitable polynucleotides are typically
from about 6 to 40 nucleotides in length. Preferably a
polynucleotide may be from about 12 to about 35 nucleotides in
length, or alternatively from about 12 to about 20 nucleotides in
length or more preferably from about 18 to about 32 nucleotides in
length. According to an alternative aspect, the polynucleotide may
be at least 40 nucleotides in length.
[0094] The inventions include pharmaceutical compositions
comprising (a) a therapeutically effect amount of a
pharmaceutically acceptable anti-connexin agent (e.g., an
anti-connexin 43 oligodeoxynucleotide) and (b) a pharmaceutically
acceptable carrier or diluent. Therapeutically effective doses and
dose concentrations are described herein. In some embodiments, the
compositions or treatment regimens of the invention include
compositions that include multiple anti-connexin agent species
(e.g., two or more anti-connexin polynucleotide species wherein
each species targets a different connexin mRNA or pre-mRNA species;
an anti-connexin polynucleotide that can target two or more
different connexin mRNA or pre-mRNA species because of conserved
sequence identity over at least a portion of the different connexin
mRNA or pre-mRNA species targeted by the anti-connexin
polynucleotide; etc.).
[0095] The anti-connexin agents of this invention may include those
agents or compounds capable of inducing phosphorylation on connexin
amino acid residues in order to induce gap junction or hemichannel
closure. Exemplary sites of phosphorylation include one or more of
a tyrosine, serine or threonine residues on the connexin protein.
In certain embodiments, modulation of phosphorylation may occur on
one or more residues on one or more connexin proteins. Exemplary
gap junction phosphorylating agents are well known in the art and
may include, for example, c-Src tyrosine kinase or other G
protein-coupled receptor agonists. See Giepmans B, J. Biol. Chem.,
Vol. 276, Issue 11, 8544-8549, Mar. 16, 2001. In one embodiment,
modulation of phosphorylation on one or more of these residues
impacts hemichannel function, particularly by closing the
hemichannel. In another embodiment, modulation of phosphorylation
on one or more of these residues impacts gap junction function,
particularly by closing the gap junction. Gap junction
phosphorylating agents that target the closure of connexin 43 gap
junctions and hemichannels are preferred.
[0096] Still other anti-connexin agents include connexin
carboxy-terminal polypeptides. See Gourdie et al.,
WO2006/069181.
[0097] In certain another aspect, gap junction modifying agent may
include, for example, aliphatic alcohols; octanol; heptanol;
anesthetics (e.g. halothane), ethrane, fluothane, propofol and
thiopental; anandamide; arylaminobenzoate (FFA: flufenamic acid and
similar derivatives that are lipophilic); carbenoxolone; Chalcone:
(2',5'-dihydroxychalcone); CHFs (Chlorohydroxyfuranones); CMCF
(3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone);
dexamethasone; doxorubicin (and other anthraquinone derivatives);
eicosanoid thromboxane A(2) (TXA(2)) mimetics; NO (nitric oxide);
Fatty acids (e.g. arachidonic acid, oleic acid and lipoxygenase
metabolites; Fenamates (flufenamic (FFA), niflumic (NFA) and
meclofenamic acids (MFA)); Genistein; glycyrrhetinic acid
(GA):18a-glycyrrhetinic acid and 18-beta-glycyrrhetinic acid, and
derivatives thereof; lindane; lysophosphatidic acid; mefloquine;
menadione; 2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin;
okadaic acid; oleamide; oleic acid; PH, gating by intracellular
acidification; e.g., acidifying agents; polyunsaturated fatty
acids; fatty acid GJIC inhibitors (e.g., oleic and arachidonic
acids); quinidine; quinine; all trans-retinoic acid; and
tamoxifen.
[0098] The anti-connexin agents of the invention, particularly
anti-connexin polynucleotides such as connexin antisense
polynucleotides (e.g., a connexin antisense oligodeooxynucleotide
such as a single-stranded anti-connexin 43 oligodeoxynucleotide),
can be used to effect treatment of a chronic skin wound by
administering to such a wound a composition that comprises about
0.001 milligram (mg) to about 10, 1, 0.1, or 0.01 mg of the
anti-connexin agent per kilogram (kg) body weight in a
pharmaceutically acceptable carrier. Preferred amounts of the
anti-connexin agent (e.g., a single-stranded anti-connexin 43
oligodeoxynucleotide) in such a composition include about 0.01 mg
to about 10 mg per kg body weight (mg/kg), about 0.01 mg to about
10 mg/kg, and about 0.5 to about 1.0 mg/kg. The dosage may also be
about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0,
4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0,
16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0,
27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0,
38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0,
49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5,
75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, or
about 100.0 mg per kg body weight, or any range or subrange between
any two of the recited doses, or any dose falling within the range
of from about 0.1 to about 100 mg per kg body weight.
[0099] In some embodiments, one or more doses of the pharmaceutical
composition may be administered at appropriate intervals. In some
aspects, the total dose (weight) of anti-connexin polynucleotide
administered to the refractory wound may be about 100 .mu.g to
about 1 mg. In some aspects, the total dose (w) of anti-connexin
polynucleotide administered may 100 .mu.g, 200 .mu.g, 300 .mu.g,
400 .mu.g, 500 .mu.g, 600 .mu.g, 700 .mu.g, 800 .mu.g, 900 .mu.g, 1
mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg,
12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21
mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, or
about 30 mg, or any amount ranging between any two of those doses.
In other embodiments, the doses will be about 10.0, 11.0, 12.0,
13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0,
24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0,
35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0,
46.0, 47.0, 48.0, 49.0, 50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0,
67.5, 70.0, 72.5, 75.0, 77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5,
95.0, 97.5, 100.0, 105, 110, 115, 120, 125, 130, 135, 140, 145,
150, 155, 160, 65, 170, 175, 180, 185, 190, 195, 200, 210, 220,
230, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or
about 500 milligrams per square centimeter, or any range or
subrange between any two of the recited doses, or any dose falling
within the range of about 1.0 to about 500 milligrams per square
centimeter.
[0100] In some aspects the dose of anti-connexin polynucleotide is
administered in a volume of about 25 ul to about 3 ml. Preferred
dose volumes for compositions that include a pharmaceutically
acceptable carrier and from 0.5 to about 1.0 mg of an anti-connexin
agent (e.g., a single-stranded anti-connexin 43
oligodeoxynucleotide) range from about 25-100 microliter (.mu.L),
from about 100-200 .mu.L, from about 200-500 .mu.L, or from about
500-1000 .mu.L.
[0101] In other embodiments, the anti-connexin agent is applied at
about a 20 .mu.M, 30 .mu.M, 40 .mu.M, 50 .mu.M, 60 .mu.M, 70 .mu.M,
80 .mu.M, 90 .mu.M, 100 .mu.M., 10-200 .mu.M, 200-300 .mu.M,
300-400 .mu.M, 400-500 .mu.M, 500-600 .mu.M, 600-700 .mu.M, 700-800
800-900 .mu.M, 900-1000 or 1000-1500 .mu.M, or 1500 .mu.M-2000
.mu.M, 2000 .mu.M-3000 .mu.M, 3000 .mu.M-4000 .mu.M, 4000
.mu.M-5000 .mu.M, 5000 .mu.M-6000 .mu.M, 6000 .mu.M-7000 .mu.M,
7000 .mu.M-8000 .mu.M, 8000 .mu.M-9000 .mu.M, 9000 .mu.M-10,000
.mu.M, 10,000 .mu.M-11,000 .mu.M, 11,000 .mu.M-12,000 .mu.M, 12,000
.mu.M-13,000 .mu.M, 13,000 .mu.M-14,000 .mu.M, 14,000 .mu.M-15,000
.mu.M, 15,000 .mu.M-20,000 .mu.M, 20,000 .mu.M-30,000 .mu.M, 30,000
.mu.M-50,000 .mu.M, or greater, or any range or subrange between
any two of the recited doses, or any dose falling within the range
of from about 20 .mu.M to about 50,000 .mu.M.
[0102] The connexin protein or proteins targeted by the
polynucleotide will be dependent upon the site at which
downregulation is to be effected. This reflects the non-uniform
make-up of gap junction(s) at different sites throughout the body
in terms of connexin sub-unit composition. The connexin is a
connexin that naturally occurs in a human or animal in one aspect
or naturally occurs in the tissue in which connexin expression or
activity is to be decreased. The connexin gene (including coding
sequence) generally has homology or nucleotide sequence identity
with the coding sequence of one or more of the specific connexins
mentioned herein. The connexin is typically an .alpha. or .beta.
connexin. Preferably the connexin is an .alpha. connexin and is
expressed in the tissue to be treated.
[0103] Some connexin proteins are, however, more ubiquitous than
others in terms of distribution in tissue. One of the most
widespread is connexin 43. Polynucleotides targeted to connexin 43
are particularly suitable for use in the present invention. In
other aspects other connexins are targeted. In one preferred
aspect, the antisense polynucleotides are targeted to the mRNA of
one connexin protein only. Most preferably, this connexin protein
is connexin 43. In another aspect, connexin protein is connexin
Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, or Cx45. In
other aspects, the connexin protein is connexin 26 or 30.
[0104] It is also contemplated that polynucleotides targeted to
separate connexin proteins be used in combination (for example 1,
2, 3, 4, or more different connexins may be targeted). For example,
polynucleotides targeted to connexin 43, and one or more other
members of the connexin family (such as connexin Cx26, Cx30,
Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, or Cx45 can be used in
combination.
[0105] Alternatively, the anticonnexin polynucleotides may be part
of compositions that may comprise polynucleotides to more than one
connexin protein. Preferably, one of the connexin proteins to which
polynucleotides are directed is connexin 43. Other connexin
proteins to which oligthe polynucleotides are directed may include,
for example, connexin Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37,
Cx40, or Cx45.
[0106] Individual anti-connexin agents may be specific to a
particular connexin, or may target 1, 2, 3, or more different
connexins. Specific polynucleotides will in some embodiments target
sequences in the targeted connexin gene, mRNA, or pre-mRNA that are
not conserved between connexins, whereas multi-specific
polynucleotides will target conserved sequences for various
connexins
[0107] The polynucleotides for use in the invention may suitably be
unmodified phosphodiester oligomers. Such oligodeoxynucleotides may
vary in length. A 30-mer polynucleotide has been found to be
particularly suitable.
[0108] Many aspects of the invention are described with reference
to oligodeoxynucleotides. However, it is understood that other
suitable polynucleotides (such as RNA polynucleotides) and
anti-connexin agents may be used in these aspects.
[0109] The anticonnexin polynucleotides may be chemically modified.
This may enhance their resistance to nucleases and may enhance
their ability to enter cells. For example, phosphorothioate
oligonucleotides may be used. Other deoxynucleotide analogs include
methylphosphonates, phosphoramidates, phosphorodithioates,
N3'P5'-phosphoramidates and oligoribonucleotide phosphorothioates
and their 2'-O-alkyl analogs and 2'-O-methylribonucleotide
methylphosphonates. Alternatively, mixed backbone oligonucleotides
("MBOs") may be used. MBOs contain segments of phosphothioate
oligodeoxynucleotides and appropriately placed segments of modified
oligodeoxy- or oligoribonucleotides. MBOs have segments of
phosphorothioate linkages and other segments of other modified
oligonucleotides, such as methylphosphonate, which is non-ionic,
and very resistant to nucleases or 2'-O-alkyloligoribonucleotides.
Methods of preparing modified backbone and mixed backbone
oligonucleotides are known in the art.
[0110] The precise sequence of representative preferred antisense
polynucleotides used in the invention will depend upon the target
connexin protein. As described, suitable connexin antisense
polynucleotides can include polynucleotides such as
oligodeoxynucleotides.
[0111] Suitable polynucleotides for the preparation of the combined
polynucleotide compositions described herein include for example,
polynucleotides to connexin 43 and polynucleotides for connexins
26, 30, 31.1, 32, and 37.
[0112] Although the precise sequence of the anticonnexin
polynucleotide used in the invention will depend upon the target
connexin protein, for connexin 43, antisense polynucleotides having
the following sequences have been found to be particularly
suitable:
TABLE-US-00001 SEQ. ID. NO: 1: 5'-GTA ATT GCG GCA AGA AGA ATT GTT
TCT GTC-3' SEQ. ID. NO: 2: 5'-GTA ATT GCG GCA GGA GGA ATT GTT TCT
GTC-3' SEQ. ID. NO: 3: 5'-GGC AAG AGA CAC CAA AGA CAC TAC CAG
CAT-3' SEQ. ID. NO: 7: 5'-GAC AGA AAC AAT TCC TCC TGC CGC ATT
TAC-3'
[0113] Suitable antisense polynucleotides for connexins 26, 31.1,
and 32 have the following sequences:
TABLE-US-00002 SEQ. ID. NO: 4 (Cx26): 5'-TCC TGA GCA ATA CCT AAC
GAA CAA ATA-3' SEQ. ID. NO: 5 (Cx31.1): 5'-CGT CCG AGC CCA GAA AGA
TGA GGT C-3' SEQ. ID. NO: 6 (Cx32): 5'-TTT CTT TTC TAT GTG CTG TTG
GTG A-3'
[0114] Polynucleotides, including ODN's, directed to connexin
proteins can be selected in terms of their nucleotide sequence by
any convenient, and conventional, approach. For example, the
computer programs MacVector and OligoTech (from Oligos etc. Eugene,
Oreg., USA) can be used. Once selected, the ODN's can be
synthesized using a DNA synthesizer.
[0115] Polynucleotide Homologues
[0116] Homology, homologues, and nucleotide sequence identity are
described herein (for example, the polynucleotide may be a
homologue of a complement to a sequence in connexin mRNA). Such a
polynucleotide typically has at least about 70% nucleotide sequence
identity, preferably at least about 80%, at least about 90%, at
least about 95%, at least about 97% or at least about 99% sequence
identity with the relevant sequence, for example, over a region of
at least about 15, at least about 20, at least about 40, at least
about 100 more contiguous nucleotides (of the homologous sequence).
Homology or sequence identity may be calculated based on any method
in the art.
[0117] For example, the BLAST algorithm performs a statistical
analysis of the similarity between two sequences; see, e.g., Karlin
and Altschul (1993), Proc. Natl. Acad. Sci. USA 90: 5873-5787. One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a sequence is
considered similar to another sequence if the smallest sum
probability in comparison of the first sequence to a second
sequence is less than about 1, preferably less than about 0.1, more
preferably less than about 0.01, and most preferably less than
about 0.001.
[0118] The homologous sequence typically differs from the relevant
sequence by at least about (or by no more than about) 2, 5, 10, 15,
20, or more mutations (which may be substitutions, deletions or
insertions). These mutations may be measured across any of the
regions mentioned above in relation to calculating sequence
identity.
[0119] The homologous sequence typically hybridizes selectively to
the original sequence at a level significantly above background.
Selective hybridization is typically achieved using conditions of
medium to high stringency (for example 0.03M sodium chloride and
0.03M sodium citrate at from about 50.degree. C. to about
60.degree. C.). However, such hybridization may be carried out
under any suitable conditions known in the art (see Sambrook, et
al. (1989), Molecular Cloning: A Laboratory Manual). For example,
if high stringency is required, suitable conditions include
0.2.times.SSC at 60.degree. C. If lower stringency is required,
suitable conditions include 2.times.SSC at 60.degree. C.
[0120] Dosage Forms and Formulations and Administration
[0121] The anti-connexin agents of the invention of the may be
administered to a subject in need of treatment, such as a subject
with any of the wounds mentioned herein. The condition of the
subject can thus be improved. The anti-connexin agent may be used
in the treatment of the subject's body by therapy. They may be used
in the manufacture of a medicament to treat any of the wounds
mentioned herein.
[0122] The anti-connexin agent (e.g., an anti-connexin
polynucleotide) may be present in a substantially isolated form. It
will be understood that the product may be mixed with carriers or
diluents that will not interfere with the intended purpose of the
product and still be regarded as substantially isolated. A product
of the invention may also be in a substantially purified form, in
which case it will generally comprise about 80%, 85%, or 90%,
including, for example, at least about 95%, at least about 98% or
at least about 99% of the polynucleotide or dry mass of the
preparation.
[0123] Depending on the intended route of administration, the
pharmaceutical products, pharmaceutical compositions, combined
preparations and medicaments of the invention may, for example,
take the form of solutions, suspensions, instillations, sprays,
salves, creams, gels, foams, ointments, emulsions, lotions, paints,
sustained release formulations, or powders, and typically contain
about 0.01% to about 1% of active ingredient(s), about 1%-50% or
active ingredient(s), about 2%-60% of active ingredient(s), about
2%-70% of active ingredient(s), or up to about 90% of active
ingredient(s). Other suitable formulations include pluronic
gel-based formulations, carboxymethylcellulose(CMC)-based
formulations, and hyroxypropylmethylcellulose(HPMC)-based
formulations. Other useful formulations include slow or delayed
release preparations.
[0124] Gels or jellies may be produced using a suitable gelling
agent including, but not limited to, gelatin, tragacanth, or a
cellulose derivative and may include glycerol as a humectant,
emollient, and preservative. Ointments are semi-solid preparations
that consist of the active ingredient incorporated into a fatty,
waxy, or synthetic base. Examples of suitable creams include, but
are not limited to, water-in-oil and oil-in-water emulsions.
Water-in-oil creams may be formulated by using a suitable
emulsifying agent with properties similar, but not limited, to
those of the fatty alcohols such as cetyl alcohol or cetostearyl
alcohol and to emulsifying wax. Oil-in-water creams may be
formulated using an emulsifying agent such as cetomacrogol
emulsifying wax. Suitable properties include the ability to modify
the viscosity of the emulsion and both physical and chemical
stability over a wide range of pH. The water soluble or miscible
cream base may contain a preservative system and may also be
buffered to maintain an acceptable physiological pH.
[0125] Foam preparations may be formulated to be delivered from a
pressurized aerosol canister, via a suitable applicator, using
inert propellants. Suitable excipients for the formulation of the
foam base include, but are not limited to, propylene glycol,
emulsifying wax, cetyl alcohol, and glyceryl stearate. Potential
preservatives include methylparaben and propylparaben.
[0126] Preferably the anti-connexin agents useful in practicing the
instant inventions are combined with a pharmaceutically acceptable
carrier or diluent to produce a pharmaceutical composition.
Suitable carriers and diluents include isotonic saline solutions,
for example phosphate-buffered saline. Suitable diluents and
excipients also include, for example, saline, dextrose, glycerol,
or the like, and combinations thereof. In addition, if desired
substances such as wetting or emulsifying agents, stabilizing or ph
buffering agents may also be present.
[0127] The term "pharmaceutically acceptable carrier" refers to any
pharmaceutical carrier that does not itself induce the production
of antibodies harmful to the individual receiving the composition,
and which can be administered without undue toxicity. Suitable
carriers can be large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, and amino acid copolymers.
[0128] Pharmaceutically acceptable salts can also be present, e.g.,
mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the
like.
[0129] The pharmaceutical carriers in the pharmaceutical
composition useful in the methods and articles of manufacture of
this disclosure may be one or more pharmaceutical carriers suitable
for topical administration. In one embodiment, the pharmaceutical
carrier may be a nonionic polyoxyethylene-polyoxypropylene
copolymer, also referred to as a poloxamer. The pharmaceutical
carrier may be present in the pharmaceutical composition at between
5 and 25-30%. For example, the pharmaceutical carrier may be
present in the pharmaceutical composition at 20% (w/w). In another
embodiment the pharmaceutical carrier may be present in the
pharmaceutical composition at about 22.0%. A preferred poloxamer is
poloxamer 407, also known as Pluronic F-127 (BASF)
[0130] The pharmaceutical compositions for use in the methods and
kits and articles of manufacture as disclosed herein may be
formulated in a delayed release preparation, a slow release
preparation, an extended release preparation, a controlled release
preparation, and/or in a repeat action preparation to a subject
with a wound characterized in whole or in part by delayed or
incomplete wound healing, or other wound that does not heal at an
expected rate. Such formulations are particularly advantageous for
wounds that do not heal at expected rates, such as chronic
wounds.
[0131] Suitable carrier materials include any carrier or vehicle
commonly used as a base for creams, lotions, sprays, foams, gels,
emulsions, lotions or paints for topical administration. Examples
include emulsifying agents, inert carriers including hydrocarbon
bases, emulsifying bases, non-toxic solvents or water-soluble
bases. Particularly suitable examples include pluronics, HPMC, CMC
and other cellulose-based ingredients, lanolin, hard paraffin,
liquid paraffin, soft yellow paraffin or soft white paraffin, white
beeswax, yellow beeswax, cetostearyl alcohol, cetyl alcohol,
dimethicones, emulsifying waxes, isopropyl myristate,
microcrystalline wax, oleyl alcohol and stearyl alcohol.
[0132] Slow release gels in which the anti-connexin agent is
released over time are preferred for topical application. Thus, in
preferred embodiments, the pharmaceutical composition may be
formulated to provide sustained release of the anti-connexin agent,
e.g., an anti-connexin antisense polynucleotide. Preferred
anti-connexin polynucleotides include anti-connexin 43
polynucleotides, particularly anti-connexin 43 antisense
polynucleotides.
[0133] Preferably, the pharmaceutically acceptable carrier or
vehicle is a gel, suitably a nonionic
polyoxyethylene-polyoxypropylene copolymer gel, for example, a
Pluronic gel, preferably Pluronic F-127 (BASF Corp.). Such a gel
can be used as a liquid at low temperatures but rapidly sets at
physiological temperatures, which can assist in confining the
release of the anti-connexin agent, particularly an anti-connexin
antisense polynucleotide (e.g., an ODN) active ingredient, to the
site of application or immediately adjacent to that site.
[0134] Other pharmaceutically acceptable carriers useful in the
articles of manufacture and methods of this disclosure include an
alginate, polyvinyl alcohol, hydrogels, including hydrogels that
contain a cellulose derivative and/or polyacrylic acid;
cellulose-based carrier, including hydroxyethyl cellulose,
hydroxymethyl cellulose, carboxymethyl cellulose,
hydroxypropylmethyl cellulose and mixtures thereof.
[0135] Other suitable formulations include pluronic gel-based
formulations, carboxymethylcellulose(CMC)-based formulations, and
hydroxypropylmethylcellulose (HPMC)-based formulations. The
composition may be formulated for any desired form of delivery,
including topical, instillation, parenteral, subcutaneous, or
transdermal administration. Other useful formulations include slow
or delayed release preparations.
[0136] The formulation that is administered may contain
transfection agents. Examples of such agents include cationic
agents (for example calcium phosphate and DEAE-dextran) and
lipofectants (for example Lipofectam.TM. and Transfectam.TM.), and
surfactants.
[0137] In some embodiments, the formulation further includes a
surfactant to assist with polynucleotide cell penetration or the
formulation may contain any suitable loading agent. Any suitable
non-toxic surfactant may be included, such as DMSO. Alternatively,
a transdermal penetration agent such as urea may be included.
[0138] In some embodiments, the effective dose for a given subject
preferably lies within the dose that is therapeutically effective
for at least 50% of the population, and that exhibits little or no
toxicity at this level.
[0139] The effective dosage of each of the anti-connexin agents
employed in the methods and compositions of the invention may vary
depending on a number of factors, including the particular
anti-connexin agent(s) employed, the mode(s) of administration, the
frequency of administration, the wound being treated, the severity
of the wound being treated, the route of administration, the needs
of a patient sub-population to be treated, or the needs of the
individual patient which different needs can be due to age, sex,
body weight, or relevant medical wound specific to the patient.
[0140] A suitable dose may be from about 0.001 mg/kg to about 10
mg/kg body weight, such as about 0.01 mg/kg to about 0.1-1.0 mg/kg
body weight. A suitable dose may, however, be from about 0.001
mg/kg to about 0.1 mg/kg body weight, such as about 0.01 mg/kg to
about 0.050 mg/kg body weight. Doses from about 1 to 100, 200, 300,
400, and 500 micrograms (.mu.g) can be used, with doses of about 30
.mu.g to about 500 pg being preferred. In other embodiments, the
doses will be about 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0,
18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0,
29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0,
40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0,
52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0, 77.5,
80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, 100.0, 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 65, 170, 175,
180, 185, 190, 195, 200, 210, 220, 230, 250, 260, 270, 280, 290,
300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,
430, 440, 450, 460, 470, 480, 490, or about 500 milligrams per
square centimeter, or any range or subrange between any two of the
recited doses, or any dose falling within the range of about 1.0 to
about 500 milligrams per square centimeter. As noted herein, repeat
applications are contemplated. Repeat applications are typically
applied about once per week, or when wound-healing may appear to be
stalled or slowing.
[0141] Still other useful dosage levels include those having
between about 1 nanogram (ng)/kg and about 1 mg/kg body weight per
day of an anti-connexin agent described herein. In certain
embodiments, the dosage of each of the subject compounds will
generally be in the range of about 1 ng/kg to about 1 .mu.g/kg body
weight, about 1 ng/kg to about 0.1 .mu.g/kg body weight, about 1
ng/kg to about 10 ng/kg body weight, about 10 ng/kg to about 0.1
.mu.g/kg body weight, about 0.1 .mu.g/kg to about 1 .mu.g/kg body
weight, about 20 ng/kg to about 100 ng/kg body weight, about 0.001
mg/kg to about 100 mg/kg body weight, about 0.01 mg/kg to about 10
mg/kg body weight, or about 0.1 mg/kg to about 1 mg/kg body weight.
In certain embodiments, the dosage of an anti-connexin agent will
generally be in the range of about 0.001 mg/kg to about 0.01 mg/kg
body weight, about 0.01 mg/kg to about 0.1 mg/kg body weight, about
0.1 mg/kg to about 1 mg/kg body weight, or about 1 mg/kg body
weight. If more than one anti-connexin agent is used, the dosage of
each anti-connexin agent need not be in the same range as the
other. For example, the dosage of one anti-connexin agent may be
between about 0.01 mg/kg to about 1 mg/kg body weight, and the
dosage of another anti-connexin agent may be between about 0.1
mg/kg to about 1 mg/kg body weight. The dosage may also be about
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0,
5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0,
17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0,
28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0,
39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0,
50.0, 52.5, 55.0, 57.5, 60.0, 62.5, 65.0, 67.5, 70.0, 72.5, 75.0,
77.5, 80.0, 82.5, 85.0, 87.5, 90.0, 92.5, 95.0, 97.5, or about
100.0 mg per kg body weight, or any range or subrange between any
two of the recited doses, or any dose falling within the range of
from about 0.1 to about 100 mg per kg body weight. As noted herein,
repeat applications are contemplated.
[0142] Other useful doses range from about 1 to about 10 .mu.g per
square centimeter (.mu.g/cm.sup.2) of wound size. Certain doses
will be about 1-2, about 1-5, about 2-4, about 5-7, and about 8-10
.mu.g/cm.sup.2 of wound size. Other useful doses are greater than
about 10 .mu.g/cm.sup.2 of wound size, including about 15
.mu.g/cm.sup.2 of wound size, about 20 .mu.g/cm.sup.2 of wound
size, about 25 .mu.g/cm.sup.2 of wound size, about 30
.mu.g/cm.sup.2 of wound size, about 35 .mu.g/cm.sup.2 of wound
size, about 40 .mu.g/cm.sup.2 of wound size, about 50
.mu.g/cm.sup.2 of wound size, and about 100 .mu.g/cm.sup.2 of wound
size. Other useful doses are about 150 .mu.g/cm.sup.2 of wound
size, about 200 .mu.g/cm.sup.2 of wound size, about 250
.mu.g/cm.sup.2 of wound size, or about 500 .mu.g/cm.sup.2 of wound
size. As noted herein, repeat applications are contemplated.
[0143] For example, in certain embodiments, the anti-connexin agent
composition may be applied at about 50 .mu.M to about 5000 .mu.M
final concentration at the treatment site and/or adjacent to the
treatment site. Preferably, the anti-connexin agent composition is
applied at about 100 .mu.M to about 3000 .mu.M final concentration,
more preferably, the anti-connexin polynucleotide composition is
applied at about 150 .mu.M to about 3000 .mu.M final concentration,
and more preferably, the anti-connexin polynucleotide composition
is applied at about 150 .mu.M to about 3300 .mu.M final
concentration. Additionally, the anti-connexin polynucleotide
composition is applied at about 150 .mu.M to about 3000 .mu.M final
concentration, and alternatively the anti-connexin polynucleotide
composition is applied at about 250 .mu.M to about 1000 .mu.M final
concentration, or at about 300 to about 1000 .mu.M final
concentration. In certain other embodiments, the anti-connexin
polynucleotide is applied at about 100 .mu.M, 200 .mu.M, 300 .mu.M,
400 .mu.M, 500 .mu.M, 600 .mu.M, 700 .mu.M, 800 .mu.M, 900 .mu.M,
1000 .mu.M, 1100 .mu.M, 1200 .mu.M, 1300 .mu.M, 1400 .mu.M, 1500
.mu.M, 1600 .mu.M, 1700 .mu.M, 1800 .mu.M, 1900 .mu.M, 2000 .mu.M,
2100 .mu.M, 2200 .mu.M, 2300 .mu.M, 2400 .mu.M, 2500 .mu.M, 2600
.mu.M, 2700 .mu.M, 2800 .mu.M, 2900 .mu.M, or about 3000 .mu.M
final concentration, or any range in between any two of these
concentrations. In other embodiments, the anti-connexin agent is
applied at about a 20 .mu.M, 30 .mu.M, 40 .mu.M, 50 .mu.M, 60
.mu.M, 70 .mu.M, 80 .mu.M, 90 .mu.M, 100 .mu.M., 10-200 .mu.M,
200-300 .mu.M, 300-400 .mu.M, 400-500 .mu.M, 500-600 .mu.M, 600-700
.mu.M, 700-800 .mu.M, 800-900 .mu.M, 900-1000 or 1000-1500 .mu.M,
or 1500 .mu.M-2000 .mu.M, 2000 .mu.M-3000 .mu.M, 3000 .mu.M-4000
.mu.M, 4000 .mu.M-5000 .mu.M, 5000 .mu.M-6000 .mu.M, 6000
.mu.M-7000 .mu.M, 7000 .mu.M-8000 .mu.M, 8000 .mu.M-9000 .mu.M,
9000 .mu.M-10,000 .mu.M, 10,000 .mu.M-11,000 .mu.M, 11,000
.mu.M-12,000 .mu.M, 12,000 .mu.M-13,000 .mu.M, 13,000 .mu.M-14,000
.mu.M, 14,000 .mu.M-15,000 .mu.M, 15,000 .mu.M-20,000 .mu.M, 20,000
.mu.M-30,000 .mu.M, 30,000 .mu.M-50,000 .mu.M, or greater, or any
range or subrange between any two of the recited doses, or any dose
falling within the range of from about 20 .mu.M to about 50,000
.mu.M.
[0144] Anti-connexin polynucleotide dose amounts include, for
example, about 0.1-1, 1-2, 2-3, 3-4, or 4-5 micrograms (.mu.g),
from about 5 to about 10 .mu.g, from about 10 to about 15 .mu.g,
from about 15 to about 20 .mu.g, from about 20 to about 30 .mu.g,
from about 30 to about 40 .mu.g, from about 40 to about 50 .mu.g,
from about 50 to about 75 .mu.g, from about 75 to about 100 .mu.g,
from about 100 .mu.g to about 250 .mu.g and from 250 .mu.g to about
500 .mu.g. Dose amounts from 0.5 to about 1.0 milligrams (mg) or
more or also provided, as noted above. Dose volumes will depend on
the size of the site to be treated, and may range, for example,
from about 25-100 microliter (.mu.L) to about 100-200 .mu.L, from
about 200-500 .mu.L to about 500-1000 .mu.L doses are also
appropriate for larger treatment sites. As noted herein, repeat
applications are contemplated.
[0145] Conveniently, the anti-connexin agent(s) is(are)
administered in a sufficient amount to downregulate expression of a
connexin protein, or modulate gap junction formation for at least
about 0.5 to 1 hour, at least about 1-2 hours, at least about 2-4
hours, at least about 4-6 hours, at least about 6-8 hours, at least
about 8-10 hours, at least about 12 hours, or at least about 24
hours post-administration.
[0146] The dosage of each of the anti-connexin agents in accordance
with the subject invention may also be determined by reference to
the concentration of the composition relative to the size, length,
depth, area, or volume of the area to which it will be applied. For
example, in certain topical and other applications, e.g.,
instillation, dosing of the pharmaceutical compositions may be
calculated based on mass (e.g. micrograms) of or the concentration
in a pharmaceutical composition (e.g. .mu.g/.mu.L) per length,
depth, area, or volume of the area of application.
[0147] The initial and any subsequent dosages administered will
depend upon factors noted herein. Depending on the oligonucleotide,
the dosage and protocol for administration will vary, and the
dosage will also depend on the method of administration selected,
for example, local or topical administration.
[0148] The doses may be administered in single or divided
applications. The doses may be administered once, or application
may be repeated.
[0149] One or more anti-connexin agents may be administered by the
same or different routes. The various agents of the invention can
be administered separately at different times during the course of
therapy, or concurrently in divided or single combination
forms.
[0150] Preferably one or more anti-connexin agents useful for wound
healing are delivered by topical administration (peripherally or
directly to a site), including but not limited to topical
administration using solid supports (such as dressings and other
matrices) and medicinal formulations (such as gels, mixtures,
suspensions and ointments). In some embodiments, the solid support
comprises a biocompatible membrane or insertion into a treatment
site. In another embodiment, the solid support comprises a dressing
or matrix. In one embodiment of the invention, the solid support
composition may be a slow release solid support composition, in
which the one or more anti-connexin agent(s) useful for wound
healing is(are) dispersed in a slow release solid matrix such as a
matrix of alginate, collagen, or a synthetic bioabsorbable polymer.
Preferably, the solid support composition is sterile or low
bio-burden. In one embodiment, a wash solution comprising one or
more anti-connexin polynucleotides can be used.
[0151] One or more doses may be administered to a subject having a
refractory wound. In some embodiments, one or more doses of the
pharmaceutical composition comprising the may be administered at
appropriate intervals. In some embodiments, the pharmaceutical
composition may be administered daily, two to six times per week,
or weekly. For example, the composition comprising the
anti-connexin agent (e.g., an anti-connexin polynucleotide) can be
administered, delivered, or otherwise exposed to the wound to
treated for an effective period of time, for example, at least
about 0.5 hours, about 1-2 hours, about 2-4 hours, about 4-6 hours,
about 6-8 hours, or for longer periods, e.g., up to 24 hours or
more. Exposures of about 1-2 hour, 2-3 hour, and 4-8 hour per
application or delivery is presently preferred. Alternatively, the
anti-connexin agent-containing compositions and formulations
described herein can be administered repeatedly, for example, once
per week until healing is seen to be proceeding or is complete, as
desired. For example, compositions of the invention may also be
applied more frequently, 2-3 times/week. They may also be applied
weekly, biweekly, or monthly. Application once or twice per week is
presently preferred. The frequency of administration and dose may
change over the course of treatment as the wound area and volume
change. In addition, further application(s) can be made in the
event wound healing once again becomes stalled or delayed.
[0152] While the delivery period will be dependent upon both the
site at which the downregulation is to be induced and the
therapeutic effect which is desired, continuous or slow-release
delivery for about 0.5 hours, about 1-2 hours, about 2-4 hours,
about 4-6 hours, about 6-8, or about 24 hours or longer is
provided. In accordance with the present invention, this may be
achieved by inclusion of the anti-connexin agent(s) in a
formulation together with a pharmaceutically acceptable carrier or
vehicle, particularly in the form of a formulation for continuous
or slow-release administration.
[0153] As noted, the one or more anti-connexin agents described may
be administered before, during, immediately following wounding, for
example, or within about 180 or more, about 120, about 90, about
60, or about 30 days, of wounding, for example.
[0154] The routes of administration and dosages described herein
are intended only as a guide since a skilled physician will
determine the optimum route of administration and dosage for any
particular patient and wound.
[0155] Any of the methods of treating a subject having or suspected
of having or a disease, disorder, and/or wound, referenced or
described herein may utilize the administration of any of the
doses, dosage forms, formulations, and/or compositions herein
described.
[0156] Wound Treatment
[0157] In instances of tissue damage (particularly with wounds
characterized by delayed healing and chronic wounds) the
formulations for use in accordance with the invention have been
found effective in both promoting the wound healing process,
reducing inflammation and in minimizing scar tissue formation. The
formulations therefore have clear benefit in the treatment of
wounds that do not heal at expected rates, whether the result of
external trauma, or disease state (such as diabetic ulcers) or
condition (such as venous ulcers, arterial ulcers, and vasculitic
ulcers) or physical processes (such as pressure ulcers).
[0158] In one aspect the invention is directed to a method of
promoting or improving wound healing in a subject suffering from or
a chronic wound, delayed healing wound or incomplete healing wound,
or other wounds that do not heal at expected rates, comprising
administration of a therapeutically effective amount of one or more
anti-connexin agents. In certain embodiments, the administration of
one or more anti-connexin agents is effective to reduce granulation
tissue deposition, promote cell migration to accelerate wound
closure and healing, to facilitate epithelial growth, or any
combination thereof.
[0159] In one aspect the invention is directed to a method of
promoting or improving wound healing in a subject, comprising
administration of one or more anti-connexin agents in an amount
effective to regulate epithelial basal cell division and growth in
a chronic wound, delayed healing wound or incomplete healing wound,
or other wound that does not heal at an expected rate. In one
embodiment, the anti-connexin agent is an anti-connexin antisense
polynucleotide effective to regulate epithelial basal cell division
and growth. In some embodiments, the anti-connexin antisense
polynucleotide is an anti-connexin 26 antisense polynucleotide, an
anti-connexin 43 antisense polynucleotide, or a mixture
thereof.
[0160] In one aspect the invention is directed to a method of
promoting or improving wound healing, comprising administration of
one or more anti-connexin agents in an amount effective to regulate
outer layer keratin secretion in a chronic wound, delayed healing
wound or incomplete healing wound, or other wound that does not
heal at an expected rate. In some embodiments, the anti-connexin
agent is an anti-connexin antisense polynucleotide effective to
regulate outer layer keratin secretion. In one embodiment, the
connexin antisense polynucleotide is an anti-connexin 43 antisense
polynucleotide, an anti-connexin 31.1 antisense polynucleotide, or
a mixture thereof.
[0161] In one aspect the invention is directed to methods of
reducing, preventing, or ameliorating tissue damage in a subject
suffering from a chronic wound, delayed healing wound or incomplete
healing wound, or other wound that does not heal at an expected
rate, comprising administration of one or more anti-connexin
agents.
[0162] In one aspect the invention is directed to sustained
administration of one or more anti-connexin agents. In some
embodiment, the anti-connexin agents are administered for at least
about 1-24 hours, at least about 0.5 hours, at least about 1 hour,
at least about 2 hours, at least about 3 hours, at least about 4
hours, at least about 5 hours, at least about 6 hours, at least
about 7 hours, at least about 8 hours, at least about 9 hours, at
least about 10 hours, at least about 11 hours, at least about 12
hours or at least about 24 hours. In some embodiments, connexin
expression is downregulated over a sustained period of time.
Preferably, connexin 43 expression is downregulated for a sustained
period of time. Conveniently, connexin 43 expression is
downregulated for at least about 0.5, 1, 2, 4, 6, 8, 10, 12, or 24
hours. Full recovery of connexin expression generally occurs within
at least about 48-72 hours following downregulation of expression.
Suitable subjects for treatment in accordance with the invention
include diabetic subjects or other subjects having a wound that
does not heal at an expected rate.
[0163] In one aspect, the present invention provides methods of
treating a subject having a chronic wound, delayed healing wound or
incomplete healing wound, or other wound that does not heal at an
expected rate, which comprises sustained administration of an
effective amount of one or more anti-connexin agents. In a further
aspect, the present invention provides methods of promoting or
improving wound healing in a subject which comprises sustained
administration of one or more anti-connexin agents to a chronic
wound, delayed healing wound or incomplete healing wound, or other
wound that does not heal at an expected rate.
[0164] According to another further aspect, the present invention
provides methods of promoting or improving wound healing in a
subject having a chronic wound, delayed healing wound or incomplete
healing wound, or other wound that does not heal at an expected
rate, which comprises sustained administration of one or more
anti-connexin agents to a wound area in an amount effective to
increase re-epithlialization rates in the wound area. In some
embodiments, such methods comprise sustained administration of an
anti-connexin 43 antisense polynucleotide, and/or an anti-connexin
31.1 antisense polynucleotide. In some embodiments, the composition
or compositions are administered in a sustained release
formulation. In other embodiments, the composition or compositions
are administered for a sustained period of time. Conveniently, the
composition is effective to decrease connexin 43 and/or 31.1 levels
or expression for at least about 24 hours. Subjects that may be
treated include diabetic subjects or other subjects having a wound
that does not heal at an expected rate.
[0165] In yet another aspect, the present invention provides
methods of promoting or improving wound healing in a subject having
a chronic wound, delayed healing wound or incomplete healing wound,
or other wound that does not heal at an expected rate, which
comprises sustained administration one or more anti-connexin agents
to a wound area in an amount effective to effective to regulate
epithelial basal cell division and growth and/or effective to
regulate outer layer keratin secretion. In one embodiment, the
composition comprises an anti-connexin antisense polynucleotide
effective to regulate epithelial basal cell division or growth,
preferably an anti-connexin 26 antisense polynucleotide, an
anti-connexin 43 antisense polynucleotide, anti-connexin 30
antisense polynucleotide or a mixture thereof, for example. In some
embodiments, the composition comprises an anti-connexin antisense
polynucleotide effective to regulate outer layer keratinization,
preferably, an anti-connexin 31.1 antisense polynucleotide. In some
embodiments, the composition or compositions are administered in a
sustained release formulation. In other embodiments, the
composition or compositions are administered for a sustained period
of time. Conveniently, the composition is effective to decrease
connexin 43, 26, and/or 30 levels or expression for at least about
24 hours. Subjects that may be treated include diabetic
subjects.
[0166] In one aspect the invention is directed to methods for
treatment or prophylaxis of skin wounds, including a refractory
chronic wound, refractory delayed healing wound or refractory
incomplete healing wound, or other refractory wound that does not
heal at an expected rate, comprising administering to a subject in
need thereof an effective amount of an anti-connexin agent
administered to said wound or a tissue associated with said wound.
In some embodiments, a composition of the present disclosure is
administered to the skin or a tissue associated with the skin of
said subject for an effective period of time. Conveniently, the
composition is effective to decrease connexin 43 levels, or block
or reduce connexin 43 hemichannel opening, for at least about 0.5
hours, about 1-2 hours, about 2-4 hours, about 4-6 hours, about 4-8
hours, about 12 hours, about 18 hours, or about 24 hours. A chronic
skin wound suitable for treatment may, for example, be selected
from the group consisting of pressure ulcers, diabetic ulcers,
venous ulcers, arterial ulcers, vasculitic ulcers, and mixed
ulcers. The chronic wound may be an arterial ulcer, which comprises
ulcerations resulting from complete or partial arterial blockage.
The chronic wound may be a venous stasis ulcer, which comprises
ulcerations resulting from a malfunction of the venous valve and
the associated vascular disease. The chronic wound may be a
trauma-induced ulcer. Subjects with other ulcers may also be
treated, including those with venous ulcers and others described
herein and known in the art.
[0167] Compositions
[0168] The present invention is directed to pharmaceutical
compositions, formulations, and their methods of manufacture and
use wherein such compositions comprise a therapeutically effective
amount of an anti-connexin agent, including, for example, an
anti-connexin polynucleotide, including anti-connexin antisense
polynucleotides. The compositions are useful in enhancing or
promoting healing of wounds that do not heal at expected rates,
including wounds that may be slow to heal or refractory to
conventional wound treatment or wound healing promoting
therapies.
[0169] In one preferred form, such compositions contain one or more
anti-connexin agent species, for example, an anti-connexin
antisense polynucleotide, to the mRNA or pre-mRNA of one connexin
protein only. Most preferably, this connexin protein is connexin
43. Alternatively, the compositions may comprise agents,
particularly polynucleotides, to more than one connexin protein.
Preferably, one of the connexin proteins to which such agents are
directed is connexin 43. Other connexin proteins to which
anti-connexin agents may be directed include, for example,
connexins 26, 30, 30.3, 31, 31.1, 32, 37, 40, and 45. Suitable
exemplary polynucleotides (and ODNs) directed to various connexins
are set forth elsewhere herein.
[0170] Many aspects of the invention are described with reference
to anti-connexin polynucleotides, particularly
oligodeoxynucleotides. However, it is understood that other
suitable polynucleotides (such as RNA polynucleotides) may be used
in these aspects. Other anti-connexin oligonucleotides are RNAi,
siRNA, and shRNA oligonucleotides.
[0171] Accordingly, in one aspect, the invention provides
compositions for use in therapeutic treatment, which comprises at
least one anti-connexin agent, preferably an anti-connexin 43
polynucleotide. In certain preferred embodiments, such composition
further comprise a pharmaceutically acceptable carrier or
vehicle.
[0172] Kits, Medicaments and Articles of Manufacturer
[0173] Optionally, one or more anti-connexin agents may also be
used in the manufacture of the medicament. In one embodiment, the
medicament comprises a therapeutically effective amount of an
anti-connexin agent, preferably an anti-connexin 43 polynucleotide,
and a pharmaceutically acceptable carrier.
[0174] As described, the kits and packages of the invention include
an article of manufacture comprising one or more containers or
vessels that contains a sufficient amount of the desired
anti-connexin agent(s) so that a therapeutically effective amount
of such agent(s) can be delivered to a patient having a delayed
healing or other chronic skin wound, for example, a chronic venous
ulcer, venous stasis ulcer, arterial ulcer, pressure ulcer,
diabetic ulcer, diabetic foot ulcer, vasculitic ulcer, decubitus
ulcer, burn ulcer, trauma-induced ulcer, infectious ulcer, mixed
ulcer, or pyoderma gangrenosum. Such kits also include instructions
for the use of such agent(s). For example, such instructions may
include directions regarding use for the treatment of a subject
having a chronic wound or a wound characterized by delayed healing.
Instructions may include instructions for use with regard to wounds
that do not heal at expected rates, such as delayed healing wounds,
incomplete healing wounds and chronic wounds. Such instructions
provide directions regarding observing the patient to be treated in
the run in period to determine if the wound is a refractory chronic
wound, and, if so, directions regarding the administration of a
therapeutically effective amount of a composition comprising the
anti-connexin agent in the kit so as to effect treatment of the
patient's wound.
[0175] In one aspect, the invention provides kits that comprise one
or more compositions or formulations described herein. For example,
the kit may include a composition comprising an effective amount of
one or more anti-connexin 43 antisense polynucleotides.
[0176] Articles of manufacture are also provided, comprising a
vessel containing a composition or formulation for use in
accordance with the invention as described herein and instructions
for use for the treatment of a subject. For example, in preferred
embodiments the article of manufacture comprises a vessel
containing an anti-connexin agent and instructions for use for the
treatment of a subject suffering from a chronic, delayed healing,
or incomplete healing wound, or other wound that does not heal at
an expected rate.
[0177] The inventions also relate to the use of anti-connexin
agents, such as anti-connexin polynucleotides, alone or in
combination with transdermal patches, dressings, bandages,
matrices, and coverings capable of being adhered or otherwise
associated with the skin of a subject. As will be appreciated, such
compositions and articles are capable of delivering a
therapeutically effective amount of one or more anti-connexin
agents, e.g., an anti-connexin polynucleotide such as an anti-Cx43
antisense ODN, to a delayed healing or chronic wound or the skin
adjacent to such a wound.
[0178] In another aspect, the invention includes articles of
manufacture comprising a vessel containing a therapeutically
effective amount of one or more anti-connexin agents and
instructions for use, including use for the treatment of a subject
having a chronic wound or a delayed or incomplete healing wound, or
other wound that does not heal at an expected rate, or a disease,
disorder, and/or condition characterized in whole or in part by a
chronic wound or delayed or incomplete wound healing, or other
wound that does not heal at an expected rate.
[0179] As noted, wound healing has been reported to be slow in
diabetes, often resulting in infection or chronic wounds that can
lead to amputation. Cell-cell communication through the gap
junction protein connexin 43 and the dynamic regulation of connexin
43 expression play pivotal roles in wound healing. In normal
tissue, such as skin, in the first 24 hours after wounding,
connexin 43 is normally downregulated and connexin 26 upregulated
in keratinocytes at the edge of the wound as they adopt a migratory
phenotype. However, in diabetic tissue, in general, and skin, in
particular, it has been found that connexin 43 is upregulated
immediately after wounding.
[0180] The examples below describe the surprising and unexpected
discovery that wounds that remain within a certain size range
(i.e., the "target size range") during a pretreatment phase can be
more effectively and efficaciously treated than wounds that more
greatly increase or decrease in size during the pretreatment phase.
In particular, target size range changes during the pretreatment
phase that provide for effective therapy surprisingly and
unexpectedly have been discovered to range from an increase in
wound size by not more than about 30% to a decrease in wound size
by not more than 35% during the pretreatment phase. Preferred
pretreatment phases range from about 1 to about 30 days, preferably
from about 5 days to about 20 days, even more preferably from about
7 days to about 14 days.
[0181] Various aspects of the invention will now be described with
reference to the following examples, which will be understood to be
provided by way of illustration only and not to constitute a
limitation on the scope of the invention.
EXAMPLES
Example 1
Positive Phase 2 Efficacy of an Anti Connexin Formulation in
Chronic Venous Leg Ulcers
[0182] This example describes the results of CoDa Therapeutics'
successful Phase 2b human clinical trial. The Phase 2b Study was a
randomized, parallel group, dose-ranging, controlled, double-blind
Phase 2b clinical study to evaluate the safety and efficacy of a
pharmaceutical formulation (Anti Connexin Formulation) comprising a
single-stranded anti-connexin 43 oligo deoxyribonucleotide in 22.6%
nonionic polyloxyethylene-polyoxypropylene copolymer (topically
applied to patients with venous leg ulcers (VLUs) over a 10-week
treatment period. The active pharmaceutical ingredient was a
Cx43asODN having the nucleotide sequence of SEQ ID NO: 1. The
primary purpose of the study was to determine if the can improve
healing efficacy for subjects having a VLU. Secondary objectives
included determining whether high or low dose Anti Connexin
Formulation is safe and tolerable for VLU patients, to identify
which Anti Connexin Formulation dose concentration is more
effective in treating patients with VLUs, and to assess the safety
and tolerability of the Anti Connexin Formulation vehicle alone (to
ascertain that vehicle alone has no negative effect on compression
bandaging, the standard of care (SOC)).
[0183] In the Phase 2b Study, 313 patients male and female patients
aged 18 years or older were enrolled at multiple sites, and
randomized in a 1:1:1:1 ratio into one of four treatment arms: low
(1.0 mg/mL) or high (3.0 mg/mL) dose Anti Connexin Formulation
treatment, including compression bandaging (standard-of-care
(SOC)); vehicle alone, in addition to SOC; or SOC alone. Once 33
subjects were randomized into the SOC-only arm, that group was
closed to recruitment and thereafter subjects were randomized on a
1:1:1 basis into the high or lose Anti Connexin Formulation or
vehicle arms of the Study. Patients in the three treatment arms
receiving Anti Connexin Formulation or vehicle plus SOC received
once-weekly applications over a 10-week treatment period or until
the first assessment of 100% VLU re-epithelialization, whichever
occurred first. Reference VLU (RVLU) complete closure was defined
as 100% re-epithelialization without drainage confirmed after two
visits, 14 days apart. For example, when a RVLU complete closure is
first observed in a particular patient (i.e., the VLU exhibits 100%
re-epithelialization), the RVLU must remain closed for at least
another 14 days in order for it to be confirmed as being completed
closed.
[0184] The primary study endpoint was assessed as the percentage
(%) change in RVLU surface area within a 10-week treatment period,
as determined by photographic planimetry. Secondary study endpoints
were: the time to RVLU complete closure within the 10-week
treatment period; incidence of RVLU complete closure within the
10-week treatment period; incidence of RVLU complete closure at
each visit within the 10-week treatment period; percentage of RVLU
surface area reduction (SAR) at each visit within the 10-week
treatment period; incidence of ulcer recurrence during the
post-treatment period; and pain in the RVLU at each visit within
the 10-week treatment period, each determined by investigator
assessment, except for pain assessment, which was assessed by the
particular patient on a categorical scale.
[0185] Safety assessment was based on the incidence of adverse
events, as determined by investigator assessment; incidence of RVLU
infection, as determined by investigator assessment; laboratory
results; and physical examinations.
[0186] The Study was broken into three periods, a 2-week screening
or "run-in" period (study visits S1, S2, and S3), a treatment
period of up to 10 weeks (study visits T0-T10), and a
post-treatment period of up to 12 weeks (study visits P0 to P5).
The screening period was designed to determine whether subjects
were eligible for the treatment phase of the Study. During the
10-week treatment period, high and low dose Anti Connexin
Formulation or vehicle plus SOC were applied weekly using dose
volumes of approximately 0.1 mL (100 uL) per 1.0 cm.sup.2 of VLU
surface area. For a given subject, treatment stopped at the first
to occur of 10 Anti Connexin Formulation or vehicle administrations
or 100% re-epithelialization of the RVLU, at which point the
subject transitioned to the post-treatment period. Subjects failing
to achieve 100% re-epithelialization of her/his RVLU at the T10
visit were exited from the Study, and contacted 30 days thereafter
to assess for serious adverse events. The post-treatment period was
intended to confirm RVLU complete closure, assess closure
durability, and to continue to monitor for serious adverse events
within 30 days after cessation of treatment. If a patient's RVLU
was not 100% closed by the P2 visit, s/he resumed treatment. If a
patient's RVLU was not 100% closed by the P3, P4, or P5 visit, s/he
was thereafter exited from the Study.
[0187] At of before the first screening period visit (S1), written
informed consent was obtained. At the S1 visit for each patient, a
Study investigator selected a single chronic RVLU, i.e., persisting
for at least 30 days prior to the S1 visit. Any RVLU clinical
diagnosis was supported by venous duplex ultrasonography
demonstrating venous reflux of greater than 0.5 seconds. The RVLU
had to be a full thickness, well-circumscribed lesion with defined
boundaries. At the end of the screening period (S3), the RVLU had
to have a clean wound base free of non-viable tissue. Each RVLU had
to be confirmed by review of wound photographs by a medical
monitor. Patients had to have an ankle brachial index of greater
than 0.80 measured at the S1 visit or within 3 months prior to the
S1 visit. Any RVLU had to have a border of at least 1.5 cm of
healthy skin between the outer edge of the wound bed and any
surrounding skin breakdown, wound, or ulcer after debridement at
the end of the S3 visit. The RVLU had to have an estimated surface
area of between 2 cm.sup.2 and 20 cm.sup.2 at the end of the S3
visit, as determined by ruler measurement (calculated using the
longest length and longest width perpendicular the longest length).
Each patient had to be able to tolerate high compression bandaging
(.about.40 mmHg at the ankle) and had to have been compliant with
standardized compression bandaging over the screening period
(visits S1-S3).
[0188] A subject was not eligible for enrollment if s/he had a
decrease or increase greater than 40% in the estimated RVLU surface
area during the 2-week screening period (to eliminate "fast
healers" or those unlikely to benefit from treatment, respectively;
more than 75% of the RVLU was on or below the malleous; the RVLU
had been treated with continuous high compression for more than one
year before the S1 visit; the RVLU wound bed had exposed bone,
tendon, or fascia; the RVLU had clinical signs of infection and or
biopsy proof of more than 10.sup.5 organisms per gram of tissue
during the screening period; the subject had cellulitis in the RVLU
leg during the screening period; the RVLU had a high volume of
exudate at the S1 visit that necessitates more than one high
compression bandage per week; or the subject has osteomyelitis.
Other standard exclusion criteria also applied, including cancer,
pregnancy or breastfeeding, recent PDGF-BB, dermal substitute, or
living skin treatment, being non-ambulatory, etc.
[0189] The results for Study are shown in FIGS. 1A-1C. These
results show when the full Study cohort is considered, a higher
than expected response rate in the vehicle-alone arm was observed.
Indeed, the results from the vehicle-alone plus SOC subjects showed
more complete wound closures and greater RVLU surface are
reductions than observed for the low dose Anti Connexin Formulation
arm. Re-analysis of the data unexpectedly revealed, in contrast to
FDA guidance, that an increase or decrease of RVLU SAR of +40% to
-40% surprisingly led to "rapid healers" (i.e., those who heal
without the need for pharmaceutical intervention) being recruited
into the study population. When a different range of RVLU SAR
increase or decrease was utilized, namely -15% to +30%, a
178-patient subpopulation was identified that exhibited
statistically significant (p<0.05) responses in the context of
the primary SAR endpoint and the complete wound closure secondary
endpoint. These results are shown in FIGS. 2, 3, and 4. This
demonstrates that patients with refractory chronic wounds had
statistically significant responses to treatment with the Anti
Connexin Formulation. Similar efficacy responses were seen with
wounds that did not decrease in size by more than about 30% (+30%)
over a standard-of-care treatment period using compression
bandaging over two weeks, and in wounds that did not decrease in
size by more than about 35% (+35%) over the two-week compression
period, in wounds that did not increase or decrease in size by more
than about -5%/+30%, -10%/+30%, -15%/+30%, -20%/+30%, 25%/+30% or
-30%/+30% over the two-week standard-of-care treatment period using
compression bandaging.
[0190] Together, the results indicate that selecting patients for
treatment with Anti Connexin Formulation benefit from pre-screening
prospective patients during a pretreatment or "run-in" phase to
identify patients whose chronic wounds, in this case VLUs, with
surface areas that remain within a pre-determined target size
range, preferably having a decrease or increase in surface are
during the "run-in" period of -15% to +30%, for example. Other
ranges are described herein and above. Patients having chronic
wounds, e.g., VLUs, that do not more greatly increase and/or
decrease in size during the "run-in" phase, from two to four weeks,
when then administered an anti-connexin composition, e.g., Anti
Connexin 43 Formulation in this case, can be expected to favorably
respond to treatment, for example, by experiencing complete wound
closure and/or by experiencing greater degrees of wound closure
than would be otherwise be expected.
[0191] All patents, publications, scientific articles, web sites,
and other documents and materials referenced or mentioned herein
are indicative of the levels of skill of those skilled in the art
to which the invention pertains, and each such referenced document
and material is hereby incorporated by reference to the same extent
as if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. The inventors (or
their assignee(s)) reserve the right to physically incorporate into
this specification any and all materials and information from any
such patents, publications, scientific articles, web sites,
electronically available information, and other referenced
materials or documents.
[0192] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. Thus, for example, in each instance
herein, in embodiments or examples of the present invention, any of
the terms "comprising", "consisting essentially of", and
"consisting of" may be replaced with either of the other two terms
in the specification. Also, the terms "comprising", "including",
"containing", etc. are to be read expansively and without
limitation.
[0193] The methods and processes illustratively described herein
suitably may be practiced in differing orders of steps, and that
they are not necessarily restricted to the orders of steps
indicated herein or in the claims. It is also understood that as
used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural reference unless the context clearly
dictates otherwise. Under no circumstances may the patent be
interpreted to be limited to the specific examples or embodiments
or methods specifically disclosed herein. Under no circumstances
may the patent be interpreted to be limited by any statement made
by any Examiner or any other official or employee of the Patent and
Trademark Office unless such statement is specifically and without
qualification or reservation expressly adopted in a responsive
writing by or on behalf of the inventors.
[0194] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
[0195] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0196] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
7130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gtaattgcgg caagaagaat tgtttctgtc
30230DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2gtaattgcgg caggaggaat tgtttctgtc
30330DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3ggcaagagac accaaagaca ctaccagcat
30427DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4tcctgagcaa tacctaacga acaaata
27525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 5cgtccgagcc cagaaagatg aggtc
25625DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6tttcttttct atgtgctgtt ggtga
25730DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 7gacagaaaca attcctcctg ccgcatttac 30
* * * * *